D-Amino Acid Derivative-Modified Peptidoglycan and Methods of Use Thereof

ABSTRACT

The present disclosure provides modified bacteria and modified peptidoglycan comprising modified D-amino acids; compositions comprising the modified bacteria or peptidoglycan; and methods of using the modified bacteria or peptidoglycan. The modified D-amino acids include a bioorthogonal functional group such as an azide, an alkyne or a norbornene group. Also provided are modified peptidoglycans conjugated to a molecule of interest via a linker.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 61/731,986, filed Nov. 30, 2012, which application isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No.R01-AI051622 awarded by the National Institute of Health. The governmenthas certain rights in the invention.

INTRODUCTION

Peptidoglycan (PG) is an essential component of the bacterial cell wall.Although experiments with organisms in vitro have yielded a wealth ofinformation on PG synthesis and maturation, it is unclear how thesestudies translate to bacteria replicating within host cells.

There is a need in the art for methods of identifying agents thatinhibit PG synthesis, e.g., in pathogenic bacteria.

SUMMARY

The present disclosure provides modified bacteria and modifiedpeptidoglycan comprising modified D-amino acids; compositions comprisingthe modified bacteria or peptidoglycan; and methods of using themodified bacteria or peptidoglycan. The modified D-amino acids include abioorthogonal functional group such as an azide, an alkyne or anorbornene group. Also provided are modified peptidoglycans conjugatedto a molecule of interest via a linker.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-E depict cell surface fluorescence resulting from incubation ofbacteria in azido- and alkynyl-D-alanine analogs followed by reactionwith click chemistry probes.

FIGS. 2A-D depict incorporation of alkyne-modified D-alanine (alkDala)into L. monocytogenes PG.

FIGS. 3A-C depict labeling of newly synthesized L. monocytogenes PG byalkDala in vitro and in vivo.

FIG. 4A-B depicts cell surface fluorescence resulting from incubation ofbacteria in norbornene-D-alanine (norDala) followed by conjugation withtetrazine-fluorescein.

DEFINITIONS

As used herein the term “isolated” is meant to describe a compound ofinterest that is in an environment different from that in which thecompound naturally occurs. “Isolated” is meant to include compounds thatare within samples that are substantially enriched for the compound ofinterest and/or in which the compound of interest is partially orsubstantially purified.

As used herein, the term “substantially purified” refers to a compoundthat is removed from its natural environment or its syntheticenvironment and is at least 60% free, at least 75% free, at least 90%free, at least 95% free, at least 98% free, or at least 99% free fromother components with which it is naturally associated, or is at least60% free, at least 75% free, at least 90% free, at least 95% free, atleast 98% free, or at least 99% free from contaminants associated withsynthesis of the compound.

The term “aryl” as used herein means 5- and 6-membered single-aromaticradicals which may include from zero to four heteroatoms. Representativearyls include phenyl, thienyl, furanyl, pyridinyl, (is)oxazoyl and thelike.

The term “lower alkyl”, alone or in combination, generally means anacyclic alkyl radical containing from 1 to about 10, e.g., from 1 toabout 8 carbon atoms, or from 1 to about 6 carbon atoms. Examples ofsuch radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl, octyl and thelike.

The term “aliphatic group” means a saturated or unsaturated linear orbranched hydrocarbon group and encompasses alkyl, alkenyl, and alkynylgroups, for example. The term “alkyl group” means a substituted orunsubstituted, saturated linear or branched hydrocarbon group or chain(e.g., C₁ to C₈) including, for example, methyl, ethyl, isopropyl,tert-butyl, heptyl, iso-propyl, n-octyl, dodecyl, octadecyl, amyl,2-ethylhexyl, and the like. Suitable substituents include carboxy,protected carboxy, amino, protected amino, halo, hydroxy, protectedhydroxy, nitro, cyano, monosubstituted amino, protected monosubstitutedamino, disubstituted amino, C₁ to C₇ alkoxy, C₁ to C₇ acyl, C₁ to C₇acyloxy, and the like. The term “substituted alkyl” means the abovedefined alkyl group substituted from one to three times by a hydroxy,protected hydroxy, amino, protected amino, cyano, halo, trifloromethyl,mono-substituted amino, di-substituted amino, lower alkoxy, loweralkylthio, carboxy, protected carboxy, or a carboxy, amino, and/orhydroxy salt. As used in conjunction with the substituents for theheteroaryl rings, the terms “substituted (cycloalkyl)alkyl” and“substituted cycloalkyl” are as defined below substituted with the samegroups as listed for a “substituted alkyl” group. The term “alkenylgroup” means an unsaturated, linear or branched hydrocarbon group withone or more carbon-carbon double bonds, such as a vinyl group. The term“alkynyl group” means an unsaturated, linear or branched hydrocarbongroup with one or more carbon-carbon triple bonds. The term “cyclicgroup” means a closed ring hydrocarbon group that is classified as analicyclic group, aromatic group, or heterocyclic group. The term“alicyclic group” means a cyclic hydrocarbon group having propertiesresembling those of aliphatic groups. The term “aromatic group” or “arylgroup” means a mono- or polycyclic aromatic hydrocarbon group, and mayinclude one or more heteroatoms, and which are further defined below.The term “heterocyclic group” means a closed ring hydrocarbon in whichone or more of the atoms in the ring are an element other than carbon(e.g., nitrogen, oxygen, sulfur, etc.), and are further defined below.

The terms “halo” and “halogen” refer to the fluoro, chloro, bromo oriodo groups. There can be one or more halogen, which are the same ordifferent.

The term “haloalkyl” refers to an alkyl group as defined above that issubstituted by one or more halogen atoms. The halogen atoms may be thesame or different. The term “dihaloalkyl” refers to an alkyl group asdescribed above that is substituted by two halo groups, which may be thesame or different. The term “trihaloalkyl” refers to an alkyl group asdescribe above that is substituted by three halo groups, which may bethe same or different. The term “perhaloalkyl” refers to a haloalkylgroup as defined above wherein each hydrogen atom in the alkyl group hasbeen replaced by a halogen atom. The term “perfluoroalkyl” refers to ahaloalkyl group as defined above wherein each hydrogen atom in the alkylgroup has been replaced by a fluoro group.

The term “cycloalkyl” means a mono-, bi-, or tricyclic saturated ringthat is fully saturated or partially unsaturated. Examples of such agroup included cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, adamantyl, cyclooctyl, cis- or trans decalin,bicyclo[2.2.1]hept-2-ene, cyclohex-1-enyl, cyclopent-1-enyl,1,4-cyclooctadienyl, and the like.

The term “(cycloalkyl)alkyl” means the above-defined alkyl groupsubstituted for one of the above cycloalkyl rings. Examples of such agroup include (cyclohexyl)methyl, 3-(cyclopropyl)-n-propyl,5-(cyclopentyl)hexyl, 6-(adamantyl)hexyl, and the like.

The term “substituted phenyl” specifies a phenyl group substituted withone or more moieties, and in some instances one, two, or three moieties,chosen from the groups consisting of halogen, hydroxy, protectedhydroxy, cyano, nitro, trifluoromethyl, C₁ to C₇ alkyl, C₁ to C₇ alkoxy,C₁ to C₇ acyl, C₁ to C₇ acyloxy, carboxy, oxycarboxy, protected carboxy,carboxymethyl, protected carboxymethyl, hydroxymethyl, protectedhydroxymethyl, amino, protected amino, (monosubstituted)amino, protected(monosubstituted)amino, (disubstituted)amino, carboxamide, protectedcarboxamide, N—(C₁ to C₆ alkyl)carboxamide, protected N—(C₁ to C₆alkyl)carboxamide, N,N-di(C₁ to C₆ alkyl)carboxamide, trifluoromethyl,N—((C₁ to C₆ alkyl)sulfonyl)amino, N-(phenylsulfonyl)amino or phenyl,substituted or unsubstituted, such that, for example, a biphenyl ornaphthyl group results.

Examples of the term “substituted phenyl” includes a mono- ordi(halo)phenyl group such as 2, 3 or 4-chlorophenyl, 2,6-dichlorophenyl,2,5-dichlorophenyl, 3,4-dichlorophenyl, 2, 3 or 4-bromophenyl,3,4-dibromophenyl, 3-chloro-4-fluorophenyl, 2, 3 or 4-fluorophenyl andthe like; a mono or di(hydroxy)phenyl group such as 2, 3, or4-hydroxyphenyl, 2,4-dihydroxyphenyl, the protected-hydroxy derivativesthereof and the like; a nitrophenyl group such as 2, 3, or4-nitrophenyl; a cyanophenyl group, for example, 2, 3 or 4-cyanophenyl;a mono- or di(alkyl)phenyl group such as 2, 3, or 4-methylphenyl,2,4-dimethylphenyl, 2, 3 or 4-(iso-propyl)phenyl, 2, 3, or4-ethylphenyl, 2, 3 or 4-(n-propyl)phenyl and the like; a mono ordi(alkoxy)phenyl group, for example, 2,6-dimethoxyphenyl, 2, 3 or4-(isopropoxy)phenyl, 2, 3 or 4-(t-butoxy)phenyl,3-ethoxy-4-methoxyphenyl and the like; 2, 3 or 4-trifluoromethylphenyl;a mono- or dicarboxyphenyl or (protected carboxy)phenyl group such as 2,3 or 4-carboxyphenyl or 2,4-di(protected carboxy)phenyl; a mono- ordi(hydroxymethyl)phenyl or (protected hydroxymethyl)phenyl such as 2, 3or 4-(protected hydroxymethyl)phenyl or 3,4-di(hydroxymethyl)phenyl; amono- or di(aminomethyl)phenyl or (protected aminomethyl)phenyl such as2, 3 or 4-(aminomethyl)phenyl or 2,4-(protected aminomethyl)phenyl; or amono- or di(N-(methylsulfonylamino))phenyl such as 2, 3 or4-(N-(methylsulfonylamino))phenyl. Also, the term “substituted phenyl”represents disubstituted phenyl groups wherein the substituents aredifferent, for example, 3-methyl-4-hydroxyphenyl,3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl,4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl,2-hydroxy-4-chlorophenyl and the like.

The term “(substituted phenyl)alkyl” means one of the above substitutedphenyl groups attached to one of the above-described alkyl groups.Examples of include such groups as 2-phenyl-1-chloroethyl,2-(4′-methoxyphenyl)ethyl, 4-(2′,6′-dihydroxy phenyl)n-hexyl,2-(5′-cyano-3′-methoxyphenyl)n-pentyl, 3-(2′,6′-dimethylphenyl)n-propyl,4-chloro-3-aminobenzyl, 6-(4′-methoxyphenyl)-3-carboxy(n-hexyl),5-(4′-aminomethylphenyl)-3-(aminomethyl)n-pentyl,5-phenyl-3-oxo-n-pent-1-yl, (4-hydroxynapth-2-yl)methyl and the like.

As noted above, the term “aromatic” or “aryl” refers to six memberedcarbocyclic rings. Also as noted above, the term “heteroaryl” denotesoptionally substituted five-membered or six-membered rings that have 1to 4 heteroatoms, such as oxygen, sulfur and/or nitrogen atoms, inparticular nitrogen, either alone or in conjunction with sulfur oroxygen ring atoms.

Furthermore, the above optionally substituted five-membered orsix-membered rings can optionally be fused to an aromatic 5-membered or6-membered ring system. For example, the rings can be optionally fusedto an aromatic 5-membered or 6-membered ring system such as a pyridineor a triazole system, e.g., to a benzene ring.

The following ring systems are examples of the heterocyclic (whethersubstituted or unsubstituted) radicals denoted by the term “heteroaryl”:thienyl, furyl, pyrrolyl, pyrrolidinyl, imidazolyl, isoxazolyl,triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl,oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, oxazinyl,triazinyl, thiadiazinyl tetrazolo, 1,5-[b]pyridazinyl and purinyl, aswell as benzo-fused derivatives, for example, benzoxazolyl,benzthiazolyl, benzimidazolyl and indolyl.

Substituents for the above optionally substituted heteroaryl rings arefrom one to three halo, trihalomethyl, amino, protected amino, aminosalts, mono-substituted amino, di-substituted amino, carboxy, protectedcarboxy, carboxylate salts, hydroxy, protected hydroxy, salts of ahydroxy group, lower alkoxy, lower alkylthio, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl, substituted(cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, and(substituted phenyl)alkyl. Substituents for the heteroaryl group are asheretofore defined, or in the case of trihalomethyl, can betrifluoromethyl, trichloromethyl, tribromomethyl, or triiodomethyl. Asused in conjunction with the above substituents for heteroaryl rings,“lower alkoxy” means a C₁ to C₄ alkoxy group, similarly, “loweralkylthio” means a C₁ to C₄ alkylthio group.

The term “(monosubstituted)amino” refers to an amino group with one substituent chosen from the group consisting of phenyl, substituted phenyl,alkyl, substituted alkyl, C₁ to C₄ acyl, C₂ to C₇ alkenyl, C₂ to C₇substituted alkenyl, C₂ to C₇ alkynyl, C₇ to C₁₆ alkylaryl, C₇ to C₁₆substituted alkylaryl and heteroaryl group. The (monosubstituted) aminocan additionally have an amino-protecting group as encompassed by theterm “protected (monosubstituted)amino.” The term “(disubstituted)amino”refers to amino groups with two substituents chosen from the groupconsisting of phenyl, substituted phenyl, alkyl, substituted alkyl, C₁to C₇ acyl, C₂ to C₇ alkenyl, C₂ to C₇ alkynyl, C₇ to C₁₆ alkylaryl, C₇to C₁₆ substituted alkylaryl and heteroaryl. The two substituents can bethe same or different.

The term “heteroaryl(alkyl)” denotes an alkyl group as defined above,substituted at any position by a heteroaryl group, as above defined.

“Optional” or “optionally” means that the subsequently described event,circumstance, feature, or element may, but need not, occur, and that thedescription includes instances where the event or circumstance occursand instances in which it does not. For example, “heterocyclo groupoptionally mono- or di-substituted with an alkyl group” means that thealkyl may, but need not, be present, and the description includessituations where the heterocyclo group is mono- or disubstituted with analkyl group and situations where the heterocyclo group is notsubstituted with the alkyl group.

Compounds that have the same molecular formula but differ in the natureor sequence of bonding of their atoms or the arrangement of their atomsin space are termed “isomers.” Isomers that differ in the arrangement oftheir atoms in space are termed “stereoisomers.” Stereoisomers that arenot minor images of one another are termed “diastereomers” and thosethat are non-superimposable mirror images of each other are termed“enantiomers.” When a compound has an asymmetric center, for example, itis bonded to four different groups, a pair of enantiomers is possible.An enantiomer can be characterized by the absolute configuration of itsasymmetric center and is described by the R- and S-sequencing rules ofCahn and Prelog, or by the manner in which the molecule rotates theplane of polarized light and designated as dextrorotatory orlevorotatory (i.e., as (+) or (−)-isomers respectively). A chiralcompound can exist as either individual enantiomer or as a mixturethereof. A mixture containing equal proportions of the enantiomers iscalled a “racemic mixture.”

The compounds of this invention may possess one or more asymmetriccenters; such compounds can therefore be produced as individual (R)— or(S)— stereoisomers or as mixtures thereof. Unless indicated otherwise,the description or naming of a particular compound in the specificationand claims is intended to include both individual enantiomers andmixtures, racemic or otherwise, thereof. The methods for thedetermination of stereochemistry and the separation of stereoisomers arewell-known in the art (see, e.g., the discussion in Chapter 4 of“Advanced Organic Chemistry”, 4th edition J. March, John Wiley and Sons,New York, 1992).

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “analkyne-modified D-amino acid” includes a plurality of such amino acidsand reference to “the azide-modified D-amino acid” includes reference toone or more azide-modified D-amino acids and equivalents thereof knownto those skilled in the art, and so forth. It is further noted that theclaims may be drafted to exclude any optional element. As such, thisstatement is intended to serve as antecedent basis for use of suchexclusive terminology as “solely,” “only” and the like in connectionwith the recitation of claim elements, or use of a “negative”limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides modified bacteria and modifiedpeptidoglycan comprising modified D-amino acids; compositions comprisingthe modified bacteria or peptidoglycan; and methods of using themodified bacteria or peptidoglycan.

Modified Peptidoglycan

The present disclosure provides modified peptidoglycan comprisingmodified D-amino acids. Modified D-amino acids allow labeling ofpeptidoglycan modified with a bioorthogonal functional group. Anyconvenient bioorthogonal functional groups may be utilized in thesubject modified D-amino acids and modified peptidoglycans.Bioorthogonal functional groups of interest include, but are not limitedto: azide, alkyne, alkene, norbornene, trans-cyclooctene and tetrazole.In some cases, the modified D-amino acids are azide-, alkyne-, ornorbornene-modified D-amino acids.

As shown schematically in FIG. 1A, peptidoglycan (PG) is a polymercomprising sugars and amino acids. The sugar component consists ofalternating residues of β-(1,4) linked N-acetylglucosamine (GlcNAc) andN-acetylmuramic acid (MurNAc). Attached to the MurNAc is a peptide chainof three to five amino acids. In some cases, a subject modified PGcomprises a peptide chain of four amino acids. In some instances, asubject modified PG comprises a peptide chain of five amino acids.

A subject modified PG comprises a peptide chain of three to five aminoacids, wherein at least one of the amino acids is a modified D-aminoacid, and wherein the peptide chain is linked to a MurNAc moiety in thePG polymer. In some instances, the at least one modified D-amino acid islocated at one or more of positions 2, 4, and 5. In some embodiments,only one amino acid of the peptide chain is modified D-amino acid, suchas an azide-, alkyne-, or norbornene-modified D-amino acid. In somecases, a subject modified PG comprises an azide-, alkyne-, ornorbornene-modified D-amino acid in position 2, i.e., the second aminoacid from the MurNAc moiety. In other cases, a subject modified PGcomprises an azide-, alkyne-, or norbornene-modified D-amino acid inposition 4, i.e., the fourth amino acid from the MurNAc moiety. In othercases, a subject modified PG comprises an azide-, alkyne-, ornorbornene-modified D-amino acid in position 5, i.e., the fifth aminoacid from the MurNAc moiety.

D-amino acids that can be modified to produce a modified D-amino acidinclude any genetically encoded or non-encoded amino acid. In someembodiments, a modified D-amino acid is any one of the twenty encodedamino acids. In some embodiments, a modified D-amino acid, such as anazide-modified, norbornene-modified or alkyne-modified D-amino acid,suitable for incorporation into PG to generate a modified PG is anon-canonical D-amino acid. In some embodiments, a modified D-aminoacid, such as an azide-modified, norbornene-modified or alkyne-modifiedD-amino acid, suitable for incorporation into PG to generate a modifiedPG is D-alanine. Diverse bacterial phyla produce and incorporate D-aminoacids other than D-alanine into PG. See, e.g., Lam et al. (2009) Science325:1552; and Cava et al. (2011) EMBO J. 30:3442. Thus, in someembodiments, a modified D-amino acid suitable for incorporation into PGto generate a modified PG is derived from D-methionine, D-leucine,D-tyrosine, D-phenylalanine, D-cysteine, or D-threonine, orD-isoleucine.

In some cases, a modified PG can comprise an azide-modified, anorbornene-modified or an alkyne-modified D-amino acid, where themodified amino acid is a non-encoded amino acid.

In some embodiments, the modified D-amino acid is described by formulaXXXI:

where L is an optional linker; and X is an azide, a norbornene, atetrazole or an alkyne.

In some embodiments, the modified D-amino acid is described by formulaXXXII:

where n is 0 or an integer from 1 to 12; Z is a linking functionalgroup; L is an optional linker; and X is an azide, a norbornene, atetrazole or an alkyne.

In certain embodiments of Formula (XXXII), n is 1, 2, 3, 4, 5 or 6. Incertain embodiments of Formula (XXXII), n is 1. In certain embodimentsof Formula (XXXII), n is 2. In certain embodiments of Formula (XXXII), nis 3. In certain embodiments of Formula (XXXII), n is 4. In certainembodiments of Formula (XXXII), n is 5. In certain embodiments ofFormula (XXXII), n is 6. In certain embodiments of Formula (XXXII), n is0.

Any convenient linking functional groups may be utilized in the subjectmodified D-amino acids. Z may be utilized in any convenientconfiguration as a linking functional group to connect the D-amino acidto the X group. In certain embodiments of Formula (XXXII), Z is selectedfrom an amido, a urethane, an ester, an ether, a thioether, asulfonamide, a keto and an amino. In certain embodiments of Formula(XXXII), Z is —NHC(O)—. In certain embodiments of Formula (XXXII), Z is—NHC(O)O—. In certain embodiments of Formula (XXXII), Z is —OCO—. Incertain embodiments of Formula (XXXII), Z is —O—. In certain embodimentsof Formula (XXXII), Z is absent.

Any convenient linkers may be utilized in the subject modified D-aminoacids. In certain embodiments, L includes a group selected from alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl amino, alkylamide, substituted alkylamide, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, L includes an alkyl or substituted alkyl group. Incertain embodiments, L includes an alkenyl or substituted alkenyl group.In certain embodiments, L includes an alkynyl or substituted alkynylgroup. In certain embodiments, L includes an alkoxy or substitutedalkoxy group. In certain embodiments, L includes an amino or substitutedamino group. In certain embodiments, L includes a carboxyl or carboxylester group. In certain embodiments, L includes an acyl amino group. Incertain embodiments, L includes an alkylamide or substituted alkylamidegroup. In certain embodiments, L includes an aryl or substituted arylgroup. In certain embodiments, L includes a heteroaryl or substitutedheteroaryl group. In certain embodiments, L includes a cycloalkyl orsubstituted cycloalkyl group. In certain embodiments, L includes aheterocyclyl or substituted heterocyclyl group.

In certain embodiments, L includes a polymer. For example, the polymermay include a polyalkylene glycol and derivatives thereof, includingpolyethylene glycol, methoxypolyethylene glycol, polyethylene glycolhomopolymers, polypropylene glycol homopolymers, copolymers of ethyleneglycol with propylene glycol (e.g., where the homopolymers andcopolymers are unsubstituted or substituted at one end with an alkylgroup), polyvinyl alcohol, polyvinyl ethyl ethers, polyvinylpyrrolidone,combinations thereof, and the like. In certain embodiments, the polymeris a polyalkylene glycol. In certain embodiments, the polymer is apolyethylene glycol. Other linkers are also possible, as shown in themodified D-amino acids and reagents described in more detail below.

Any convenient X groups may be utilized in the subject D-modified aminoacids. X may be connected to the amino acid via optional linker L viaany convenient configuration and at any convenient position of X. Incertain embodiments of Formula (XXXII), X is an azide-containing group.In certain embodiments of Formula (XXXII), X is —N₃.

In certain embodiments of Formula (XXXII), X is norbornene.

In certain embodiments of Formula (XXXII), X is tetrazole.

In certain embodiments of Formula (XXXII), X is an alkyne. In certainembodiments of Formula (XXXII), X is a propargyl group.

In certain embodiments of Formula (XXXII), X is a cyclooctyne. Anyconvenient cyclooctyne groups may be utilized in the subject D-aminoacids. Cyclooctyne groups of interest which may be adapted for use inthe subject modified D-amino acids include, but are not limited to, oneof the cyclooctyne groups described in the azide-reactive reagentsherein. In certain embodiments, the modified D-amino acid includes acyclooctyne groups (e.g., X) as described in one of Formulae Ito XXVIherein.

In certain embodiments of Formula (XXXII), X is described by thefollowing structure:

In certain embodiments of Formula (XXXII), X is described by thefollowing structure:

In certain embodiments of Formula (XXXII), X is described by one of thefollowing structures:

In certain embodiments of Formula (XXXII), Z is —NHC(O)O— and X-L isselected from one of the following:

In certain embodiments, n is 1. In certain embodiments, n is 4.

In certain embodiments, the modified D-amino acid isR-2-amino-3-azidopropanoic acid or propargylglycine. In someembodiments, the modified D-amino acid is described by one of thefollowing:

Analogs of such peptides include those containing residues other thannaturally occurring L-amino acids, e.g., D-amino acids or non-naturallyoccurring synthetic amino acids (e.g., non-natural, non-encoded aminoacids).

The following are non-limiting examples of amino acid modifications(e.g., modifications to introduce a bioorthogonal functional group suchas an azide, an alkyne, a norbornene, a trans-cyclooctene, or atetrazole) that can be made:

a) substitution of alkyl-substituted hydrophobic amino acids: includingalanine, leucine, isoleucine, valine, norleucine, (S)-2-aminobutyricacid, (S)-cyclohexylalanine or other simple alpha-amino acidssubstituted by an aliphatic side chain from C₁-C₁₀ carbons includingbranched, cyclic and straight chain alkyl, alkenyl or alkynylsubstitutions;

b) substitution of aromatic-substituted hydrophobic amino acids:including phenylalanine, tryptophan, tyrosine, sulfotyrosine,biphenylalanine, 1-naphthylalanine, 2-naphthylalanine,2-benzothienylalanine, 3-benzothienylalanine, histidine, includingamino, alkylamino, dialkylamino, aza, halogenated (fluoro, chloro,bromo, or iodo) or alkoxy (from C₁-C₄)-substituted forms of theabove-listed aromatic amino acids, illustrative examples of which are:2-, 3- or 4-aminophenylalanine, 2-, 3- or 4-chlorophenylalanine, 2-, 3-or 4-methylphenylalanine, 2-, 3- or 4-methoxyphenylalanine, 5-amino-,5-chloro-, 5-methyl- or 5-methoxytryptophan, 2′-, 3′-, or 4′-amino-,2′-, 3′-, or 4′-chloro-, 2, 3, or 4-biphenylalanine, 2′-, 3′-, or4′-methyl-, 2-, 3- or 4-biphenylalanine, and 2- or 3-pyridylalanine;

c) substitution of amino acids containing basic side chains: includingarginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid,homoarginine, including alkyl, alkenyl, or aryl-substituted (from C₁-C₁₀branched, linear, or cyclic) derivatives of the previous amino acids,whether the substituent is on the heteroatoms (such as the alphanitrogen, or the distal nitrogen or nitrogens, or on the alpha carbon,in the pro-R position for example. Compounds that serve as illustrativeexamples include: N-epsilon-isopropyl-lysine,3-(4-tetrahydropyridyl)-glycine, 3-(4-tetrahydropyridyl)-alanine,N,N-gamma,gamma′-diethyl-homoarginine. Included also are compounds suchas alpha-methyl-arginine, alpha-methyl-2,3-diaminopropionic acid,alpha-methyl-histidine, alpha-methyl-ornithine where the alkyl groupoccupies the pro-R position of the alpha-carbon. Also included are theamides formed from alkyl, aromatic, heteroaromatic (where theheteroaromatic group has one or more nitrogens, oxygens or sulfur atomssingly or in combination) carboxylic acids or any of the many well-knownactivated derivatives such as acid chlorides, active esters, activeazolides and related derivatives) and lysine, ornithine, or2,3-diaminopropionic acid;

d) substitution of acidic amino acids: including aspartic acid, glutamicacid, homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl, andheteroaryl sulfonamides of 2,4-diaminopriopionic acid, ornithine orlysine and tetrazole-substituted alkyl amino acids;

e) substitution of side chain amide residues: including asparagine,glutamine, and alkyl or aromatic substituted derivatives of asparagineor glutamine; and

f) substitution of hydroxyl containing amino acids: including serine,threonine, homoserine, 2,3-diaminopropionic acid, and alkyl or aromaticsubstituted derivatives of serine or threonine.

In some cases, a subject modified PG comprises one or more naturallyoccurring non-genetically encoded L-amino acids, synthetic L-amino acidsor D-enantiomers of an amino acid. A “non-naturally encoded amino acid”refers to an amino acid that is not one of the 20 common amino acids orpyrrolysine or selenocysteine. Other terms that may be used synonymouslywith the term “non-naturally encoded amino acid” are “non-natural aminoacid,” “unnatural amino acid,” “non-naturally-occurring amino acid,” andvariously hyphenated and non-hyphenated versions thereof.

Reagents

The modified PG or modified D-amino acid may be conjugated to anyconvenient reagent, such as a reagent that includes a detectable label.The reagent may include a compatible functional group that is capable ofconjugating to the modified PG, e.g., via bioorthogonal conjugation withthe bioorthogonal functional group (e.g., an azide, a norbornene oralkyne group) of a modified PG. A variety of bioorthogonal chemistriesand reagents may be utilized in the subject modified PGs, D-amino acidsand conjugation reagents, including but not limited to, Click chemistrygroups and reagents (e.g., as described by Sharpless et al., (2001),“Click Chemistry: Diverse Chemical Function from a Few Good Reactions”,Angewandte Chemie International Edition 40 (11): 2004-2021),photoinduced cycloaddition chemistry groups and reagents (e.g.,photo-click chemistry and groups, as described by Lin et al.,“Photoinducible Bioorthogonal Chemistry: A Spatiotemporally ControllableTool to Visualize and Perturb Proteins in Live Cells”, ACCOUNTS OFCHEMICAL RESEARCH ‘828-839’ 2011 ‘Vol. 44, No. 9); norbornene-tetrazinechemistry groups and reagents (e.g., as described by Carell et al. “AGenetically Encoded Norbornene Amino Acid for the Mild and SelectiveModification of Proteins in a Copper-Free Click Reaction”, Angew. Chem.Int. Ed. 1012, 51, 4466-4469); Staudinger ligation groups and reagents(e.g., as described by Bertozzi et al., (2000), “Cell SurfaceEngineering by a Modified Staudinger Reaction”, Science 287 (5460):2007) (e.g., using azido and phosphine groups), and other bioconjugationgroups and reagents (e.g., as described by Hermanson, BioconjugateTechniques, Second Edition, Academic Press, 2008). In certainembodiments, the reagent includes a compatible functional group selectedfrom an azido, a phosphine (e.g., a triaryl phosphine or a trialkylphosphine or mixtures thereof), a dithiol, an active ester, an alkynyl,an alkenyl, a tetrazine, a tetrazole, a hydrazoyl chloride, and anorbornenyl.

Reagents Reactive with Azide-Modified D-Amino Acids

Any convenient azide-reactive reagents may be utilized for conjugationto the subject azide-modified PGs and D-amino acids. Reagents reactivewith azide-modified amino acids include, but are not limited to,reagents that include an alkyne functional group, such as clickchemistry or copper-free click chemistry reagents.

Alkynyl Reagents

Any convenient alkynyl groups and chemistries may be adapted for use inthe subject azido-reactive reagents. Alkynyl groups and chemistries ofinterest include, but are not limited to: the cycloalkyne andheterocycloalkyne groups described by Bertozzi et al. in U.S. patentapplication Ser. No. 12/049,034, filed Mar. 14, 2008; the modifiedcycloalkyne groups described by Jewett et al. in U.S. patent applicationSer. No. 13/024,908, filed Feb. 10, 2011; the fused cyclooctynecompounds described by Van Delft et al. in WO/2011/136645, whichapplications are incorporated herein by reference in their entirety.

In some embodiments, the alkynyl reagent is a compound of the formula:

X-L-Y, wherein:

X is an alkyne group, optionally substituted with Y, and in someembodiments one or more additional groups; L is a linker; and Y is amolecule of interest, e.g., a detectable label.

In some cases, L is (T)_(n), where each T is a divalent moietyindependently selected from alkylene, substituted alkylene, alkenylene,substituted alkenylene, alkynylene, substituted alkynylene, arylene,substituted arylene, cycloalkylene, substituted cycloalkylene,heteroarylene, substituted heteroarylene, heterocyclene, substitutedheterocyclene, acyl, amido, acyloxy, urethanylene, thioester, sulfonyl,sulfonamide, sulfonyl ester, —O—, —S—, —NH—, and substituted amine; andeach n is a number selected from zero to 40.

In some embodiments, Y is a molecule of interest, where suitablemolecules of interest include, but are not limited to, a detectablelabel; a toxin (including cytotoxins); a peptide; a drug; a member of aspecific binding pair; an epitope tag; a strained azacycloalkynonegroup; and the like.

In some embodiments, X is a cycloalkyne group (e.g., a cyclooctynegroup) or a heterocycloalkyne group.

In some embodiments, the alkynyl reagent is described by Formula I:

where:

Y is a molecule of interest; and

R₁ is a linker (e.g., as described herein) that includes one or more ofthe following groups: carboxylic acid, an alkyl ester, an aryl ester, asubstituted aryl ester, an aldehyde, an amide, an aryl amide, an alkylhalide, a thioester, a sulfonyl ester, an alkyl ketone, an aryl ketone,a substituted aryl ketone, a halosulfonyl, a nitrile, and a nitro. R₁can be at any position on the cyclooctyne group other than at the twocarbons joined by the triple bond.

In some instances, a cyclooctyne compound that may be adapted for use inthe subject alkynyl reagents, is described by:

In some embodiments, the alkynyl reagent is of Formula I, wherein one ormore of the carbon atoms in the cyclooctyne ring, other than the twocarbon atoms joined by a triple bond, is substituted with one or moreelectron-withdrawing groups, e.g., a halo (bromo, chloro, fluoro, iodo),a nitro group, a cyano group, a sulfone group, or a sulfonic acid group.Thus, e.g., in some embodiments, a subject alkynyl reagent is of FormulaII:

where: each of X and X′ is independently:

(a) H;

(b) one or two halogen atoms (e.g., bromo, chloro, fluoro, iodo);

(c) —W—(CH₂)_(n)—Z (where: n is an integer from 1-4 (e.g., n=1, 2, 3, or4); W, if present, is O, N, or S; and Z is nitro, cyano, sulfonic acid,or a halogen);

(d) —(CH₂)_(n)—W—(CH₂)_(m)—Z (where: n and m are each independently 1 or2; W is O, N, S, or sulfonyl; if W is O, N, or S, then Z is nitro,cyano, or halogen; and if W is sulfonyl, then Z is H); or

(e) —(CH₂)_(n)—Z (where: n is an integer from 1-4 (e.g., n=1, 2, 3, or4); and Z is nitro, cyano, sulfonic acid, or a halogen);

Y is a molecule of interest; and

R₁ is a linker that includes one or more of the following groups:carboxylic acid, an alkyl ester, an aryl ester, a substituted arylester, an aldehyde, an amide, an aryl amide, an alkyl halide, athioester, a sulfonyl ester, an alkyl ketone, an aryl ketone, asubstituted aryl ketone, a halosulfonyl, a nitrile, and a nitro. R₁ canbe at any position on the cyclooctyne group other than at the twocarbons linked by the triple bond.

In some instances, a cyclooctyne compound that may be adapted for use inthe subject alkynyl reagents, is described by:

In some embodiments, the alkynyl reagent is of Formula III:

wherein each of R₁-R₆ is independently H; one or two halogen atoms(e.g., bromo, chloro, fluoro, iodo); a carboxylic acid; an alkyl ester;an aryl ester; a substituted aryl ester; an aldehyde; an amine; a thiol;an amide; an aryl amide; an alkyl halide; a thioester; a sulfonyl ester;an alkyl ketone; an aryl ketone; a substituted aryl ketone; ahalosulfonyl; a nitrile; a nitro; —W—(CH₂)_(n)—Z (where: n is an integerfrom 1-4 (e.g., n=1, 2, 3, or 4), wherein W, if present, is O, N, or S;and Z is nitro, cyano, sulfonic acid, or a halogen);—(CH₂)_(n)—W—(CH₂)_(m)—Z (where: n and m are each independently 1 or 2;W is O, N, S, or sulfonyl, wherein if W is O, N, or S, then Z is nitro,cyano, or halogen, and wherein and if W is sulfonyl, then Z is H); or—(CH₂)_(n)—Z (where: n is an integer from 1-4 (e.g., n=1, 2, 3, or 4),and wherein Z is nitro, cyano, sulfonic acid, or a halogen);

wherein R₃ is optionally linked to R₄ through Y thus forming asubstituted or unsubstituted cycloalkyl or substituted or unsubstitutedheterocycloalkyl substituient on the cycloalkyne ring, wherein Y, ifpresent, is C, O, N, or S; and

wherein each of R₁-R₆ is optionally independently linked to a moietythat comprises a reactive group that facilitates covalent attachment ofa molecule of interest; or a molecule of interest.

In some embodiments of Formula III, R₁ is two fluoride atoms, one ormore of R₂, R₃, R₄, and R₅ is a fluorophore, and R₆ is —OR₇, where —OR₇is a leaving group with a quencher (e.g., and ester, a sulfonate, etc.).

In some instances, a cyclooctyne compound that may be adapted for use inthe subject alkynyl reagents, is described by:

where R₈ is selected from H; a halogen atom (e.g., bromo, fluoro,chloro, iodo); an aliphatic group, a substituted or unsubstituted alkylgroup; an alkenyl group; an alkynyl group; a carboxylic acid, an alkylester; an aryl ester; a substituted aryl ester; an aldehyde; an amine; athiol; an amide; an aryl amide; an alkyl halide; a thioester; a sulfonylester; an alkyl ketone; an aryl ketone; a substituted aryl ketone; ahalosulfonyl; a nitrile; and a nitro.

In some embodiments, the alkynyl reagent is of Formula IV:

where:

Y is a molecule of interest; and

R₁ is a linker that includes one or more of the following groups: acarboxylic acid, an alkyl ester, an aryl ester, a substituted arylester, an aldehyde, an amide, an aryl amide, an alkyl halide, athioester, a sulfonyl ester, an alkyl ketone, an aryl ketone, asubstituted aryl ketone, a halosulfonyl, a nitrile, and a nitro. R₁ canbe at any position on the cyclooctyne group other than at the twocarbons linked by the triple bond.

In some instances, a cyclooctyne compound that may be adapted for use inthe subject alkynyl reagents, is described by:

In some embodiments, the alkynyl reagent is of Formula V:

where:

Y is a molecule of interest; and

R₁ is a linker that includes one or more of the following groups:carboxylic acid, an alkyl ester, an aryl ester, a substituted arylester, an aldehyde, an amide, an aryl amide, an alkyl halide, athioester, a sulfonyl ester, an alkyl ketone, an aryl ketone, asubstituted aryl ketone, a halosulfonyl, a nitrile, and a nitro. R₁ canbe at any position on the cyclooctyne group other than at the twocarbons linked by the triple bond, and other than thefluoride-substituted carbon.

In some instances, a cyclooctyne compound that may be adapted for use inthe subject alkynyl reagents, is described by one of the following:

In some embodiments, the alkynyl reagent is of Formula VI:

where Y is a molecule of interest; and

R₁ is a linker that includes one or more of the following groups: acarboxylic acid, an alkyl ester, an aryl ester, a substituted arylester, an aldehyde, an amide, an aryl amide, an alkyl halide, athioester, a sulfonyl ester, an alkyl ketone, an aryl ketone, asubstituted aryl ketone, a halosulfonyl, a nitrile, and a nitro. R₁ canbe at any position on the cyclooctyne group other than at the twocarbons linked by the triple bond, and other than thefluoride-substituted carbons.

In some embodiments, the alkynyl reagent is of Formula VII:

wherein five of X₁-X₆ are carbon;

wherein one of X₁-X₆ is N, O, P, or S;

wherein each of R₁-R₆ is independently H; one or two halogen atoms(e.g., bromo, chloro, fluoro, iodo); a carboxylic acid; an alkyl ester;an aryl ester; a substituted aryl ester; an aldehyde; an amine; a thiol;an amide; an aryl amide; an alkyl halide; a thioester; a sulfonyl ester;an alkyl ketone; an aryl ketone; a substituted aryl ketone; ahalosulfonyl; a nitrile; a nitro; —W—(CH₂)_(n)—Z (where: n is an integerfrom 1-4 (e.g., n=1, 2, 3, or 4), wherein W, if present, is O, N, or S;and Z is nitro, cyano, sulfonic acid, or a halogen);—(CH₂)_(n)—W—(CH₂)_(m)—Z (where: n and m are each independently 1 or 2;W is O, N, S, or sulfonyl, wherein if W is O, N, or S, then Z is nitro,cyano, or halogen, and wherein and if W is sulfonyl, then Z is H); or—(CH₂)_(n)—Z (where: n is an integer from 1-4 (e.g., n=1, 2, 3, or 4),and wherein Z is nitro, cyano, sulfonic acid, or a halogen); and

wherein each of R₁-R₆ is optionally independently linked to a moleculeof interest.

In some embodiments, a cycloctyne compound that may be adapted for usein the alkynyl reagent is described by one of Formulas VIII and IX:

where each —OR is independently a leaving group.

In some embodiments, a subject modified cycloalkyne is a heteroalkyne ofFormula X:

wherein each of R₁-R₆ is independently H; one or two halogen atoms(e.g., bromo, chloro, fluoro, iodo); a carboxylic acid; a methoxy group;an alkyl ester; an aryl ester; a substituted aryl ester; an aldehyde; anamine; a thiol; an amide; an aryl amide; an alkyl halide; a thioester; asulfonyl ester; an alkyl ketone; an aryl ketone; a substituted arylketone; a halosulfonyl; a nitrile; a nitro; —W—(CH₂)_(n)—Z (where: n isan integer from 1-4 (e.g., n=1, 2, 3, or 4), wherein W, if present, isO, N, or S; and Z is nitro, cyano, sulfonic acid, or a halogen);—(CH₂)_(n)—W—(CH₂)_(m)—Z (where: n and m are each independently 1 or 2;W is O, N, S, or sulfonyl, wherein if W is O, N, or S, then Z is nitro,cyano, or halogen, and wherein and if W is sulfonyl, then Z is H); or—(CH₂)_(n)—Z (where: n is an integer from 1-4 (e.g., n=1, 2, 3, or 4),and wherein Z is nitro, cyano, sulfonic acid, or a halogen); and

wherein each of R₁-R₆ is optionally independently linked to a moleculeof interest.

In some instances, a cyclooctyne compound that may be adapted for use inthe subject alkynyl reagents, is described by one of the following:

In some embodiments, the alkynyl reagent has the structure of one ofFormulas XI-XVI:

where R, R₁, R₂, and R₃ are each independently H; one or two halogenatoms (e.g., bromo, chloro, fluoro, iodo); a carboxylic acid; a methoxygroup; an alkyl ester; an aryl ester; a substituted aryl ester; analdehyde; an amine; a thiol; an amide; an aryl amide; an alkyl halide; athioester; a sulfonyl ester; an alkyl ketone; an aryl ketone; asubstituted aryl ketone; a halosulfonyl; a nitrile; or a nitro;

wherein —OR is in some embodiments a leaving group with a quencher; and

wherein each R is optionally independently linked to a molecule ofinterest.

In some embodiments, the alkynyl reagent has the structure of FormulaXVII:

where R₁ and R₂ are each independently H; one or two halogen atoms(e.g., bromo, chloro, fluoro, iodo); a carboxylic acid; an alkyl ester;an aryl ester; a substituted aryl ester; an aldehyde; an amine; a thiol;an amide; an aryl amide; an alkyl halide; a thioester; a sulfonyl ester;an alkyl ketone; an aryl ketone; a substituted aryl ketone; ahalosulfonyl; a nitrile; or a nitro; and

wherein each of R₁ and R₂ is optionally independently linked to a amoiety that comprises a reactive group that facilitates covalentattachment of a molecule of interest; or a molecule of interest.

In some instances, a cyclooctyne compound that may be adapted for use inthe subject alkynyl reagents, is described by:

In some embodiments, the alkynyl reagent has the structure of FormulaXVIII:

where Z is —CH₂—CH₂—, —CH═CH—, —Se(O)O—, —C(O)O—, —C(R₃)(R₄)O—,—N(R₅)N(R₆)—, —CH(OR₇)CH₂—, or —S(O)O—;

where R₁ and R₂ are each independently H; one or two halogen atoms(e.g., bromo, chloro, fluoro, iodo); a carboxylic acid; an alkyl ester;an aryl ester; a substituted aryl ester; an aldehyde; an amine; a thiol;an amide; an aryl amide; an alkyl halide; a thioester; a sulfonyl ester;an alkyl ketone; an aryl ketone; a substituted aryl ketone; ahalosulfonyl; a nitrile; or a nitro;

where R₃ to R₇ is each independently selected from H; a halogen atom(e.g., bromo, fluoro, chloro, iodo); an aliphatic group, a substitutedor unsubstituted alkyl group; an alkenyl group; an alkynyl group; acarboxylic acid, an alkyl ester; an aryl ester; a substituted arylester; an aldehyde; an amine; a thiol; an amide; an aryl amide; an alkylhalide; a thioester; a sulfonyl ester; an alkyl ketone; an aryl ketone;a substituted aryl ketone; a halosulfonyl; a nitrile; or a nitro; and

wherein each of R₁ and R₂ is optionally independently linked to amolecule of interest.

In some embodiments, the alkynyl reagent is described by the structureof one of Formulas XIX, XX, and XXI:

where R₁ and R₂ are as defined above for Formula XVIII.

In some embodiments, the alkynyl reagent is described by the structureof Formula XXIb:

wherein R1 and R2 are each independently H; one or two halogen atoms(e.g., bromo, chloro, fluoro, iodo); a carboxylic acid; an alkyl ester;an aryl ester; a substituted aryl ester; an aldehyde; an amine; a thiol;an amide; an aryl amide; an alkyl halide; a thioester; a sulfonyl ester;an alkyl ketone; an aryl ketone; a substituted aryl ketone; ahalosulfonyl; a nitrile; or a nitro;

wherein each n is independently 0, 1, or 2; and

wherein each of R₁ and R₂ is optionally independently linked to a amolecule of interest.

In some embodiments, the alkynyl reagent is of Formula XXIII:

wherein

five of X¹-X⁶ are carbons, where X¹-X⁶ may be saturated or unsaturated,substituted or unsubstituted;

one of X¹-X⁶ is nitrogen;

the X¹-X⁶ that is vicinal to the X¹-X⁶ that is nitrogen is C═O;

at least two of X¹-X⁶ are sp² centers vicinal to each other;

each L is a divalent moiety independently selected from alkylene,substituted alkylene, alkenylene, substituted alkenylene, alkynylene,substituted alkynylene, arylene, substituted arylene, cycloalkylene,substituted cycloalkylene, heteroarylene, substituted heteroarylene,heterocyclene, substituted heterocyclene, acyl, amido, acyloxy,urethanylene, thioester, sulfonyl, sulfonamide, sulfonyl ester, —O—,—S—, —NH—, and substituted amine;

each n is a number selected from zero to 40; and

Y is a molecule of interest.

In some embodiments, in Formula XXII, one of X²-X⁵ is nitrogen. In someembodiments, in Formula XXII, one of X³ and X⁴ is nitrogen. In certainembodiments, two of X¹-X⁶ are cyclically linked to form a fused arylring. In certain cases, X⁵ and X⁶ are part of a fused phenyl ring,and/or X¹ and X² are part of a fused phenyl ring

In some embodiments, in Formula XXII, at least four of X¹-X⁶ are sp²centers vicinal to each other. In some embodiments, in Formula XXII, X¹and X² are sp² centers vicinal to each other. In some embodiments, inFormula XXII, X⁵ and X⁶ are sp² centers vicinal to each other. In someembodiments, in Formula XXII, X² and X³ are sp² centers vicinal to eachother.

In some embodiments, in Formula XXII, at least one of X¹-X² is thecarbon of a carbonyl group. In some embodiments, in Formula XXII, atleast one of X³ and X⁴ is the carbon of a carbonyl group. In someembodiments, in Formula XXII, at least one of X⁴ and X⁵ is the carbon ofa carbonyl group. In some embodiments, the carbonyl group is thecarbonyl of an amido group. In some embodiments, the carbonyl group isthe carbonyl of an urea group.

In some embodiments, the alkynyl reagent is of Formula XXIII:

wherein

at least two of X¹, X², X⁵, and X⁶ are sp² centers vicinal to each other(e.g., X¹ and X², and/or X⁵ and X⁶), where X¹ and X², may be cyclicallylinked (e.g., to form a fused a phenyl ring) and X⁵ and X⁶, may becyclically linked (e.g., to form a fused a phenyl ring);

each L is a divalent moiety independently selected from alkylene,substituted alkylene, alkenylene, substituted alkenylene, alkynylene,substituted alkynylene, arylene, substituted arylene, cycloalkylene,substituted cycloalkylene, heteroarylene, substituted heteroarylene,heterocyclene, substituted heterocyclene, acyl, amido, acyloxy,urethanylene, thioester, sulfonyl, sulfonamide, sulfonyl ester, —O—,—S—, —NH—, and substituted amine;

n is a integer selected from zero to 40; and

Y is a molecule of interest.

In some embodiments, the alkynyl reagent is of Formula XXIV:

wherein

L is a divalent moiety selected from alkylene, substituted alkylene,alkenylene, substituted alkenylene, alkynylene, substituted alkynylene,arylene, substituted arylene, cycloalkylene, substituted cycloalkylene,heteroarylene, substituted heteroarylene, heterocyclene, substitutedheterocyclene, acyl, amido, acyloxy, urethanylene, thioester, sulfonyl,sulfonamide, sulfonyl ester, —O—, —S—, —NH—, and substituted amine;

n is an integer selected from zero to 40;

each R is independently selected from alkyl, substituted alkyl, alkoxy,substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,thioketo, carboxyl, carboxylalkyl, thiol, thioalkoxy, substitutedthioalkoxy, aryl, aryloxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl;

each a is an integer selected from zero to four; and

Y is a molecule of interest.

In Formula XXIV, the —(R)a may represent one or more optional arylsubstituents (e.g., 1, 2, 3 or 4 aryl substituents), each R groupindependently attached to any suitable carbon of the aryl ring.

In some embodiments, the alkynyl reagent is of Formula XXV:

where

X¹-X⁸ are each independently selected from carbon (e.g., CH or CR),nitrogen and silicon (e.g., Si—R);

each L is a divalent moiety selected from alkylene, substitutedalkylene, alkenylene, substituted alkenylene, alkynylene, substitutedalkynylene, arylene, substituted arylene, cycloalkylene, substitutedcycloalkylene, heteroarylene, substituted heteroarylene, heterocyclene,substituted heterocyclene, acyl, amido, acyloxy, urethanylene,thioester, sulfonyl, sulfonamide, sulfonyl ester, —O—, —S—, —NH—, and

substituted amine number independently selected from zero to 40;

each R is independently selected from alkyl, substituted alkyl, alkoxy,substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,thioketo, carboxyl, carboxylalkyl, thiol, thioalkoxy, substitutedthioalkoxy, aryl, aryloxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl; and

Y¹—Y³ are each independently selected from H and a molecule of interest.

In some embodiments, the alkynyl reagent is of Formula XXVI:

wherein

L is a divalent moiety selected from alkylene, substituted alkylene,alkenylene, substituted alkenylene, alkynylene, substituted alkynylene,arylene, substituted arylene, cycloalkylene, substituted cycloalkylene,heteroarylene, substituted heteroarylene, heterocyclene, substitutedheterocyclene, acyl, amido, acyloxy, urethanylene, thioester, sulfonyl,sulfonamide, sulfonyl ester, —O—, —S—, —NH—, and substituted amine;

n is an integer selected from zero to 40; and

Y is a molecule of interest.

In some embodiments, the alkynyl reagent, or a precursor thereof, isdescribed by the structure of one of the following:

wherein R is selected from hydrogen, alkyl, sulfate, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclyl, substituted heterocyclyl,sulfonyl, sulfonamide, sulfonyl ester, amino, and substituted amino;

A subject alkynyl reagent may be prepared using any convenient method,including but not limited to coupling a molecule of interest to acyclooctyne group (e.g., as described herein) using any suitable methodand chemistry. For example, coupling can be achieved using ametal-catalyzed cross-coupling or metal-halogen exchange/nucleophilicattack method, as illustrated in the following scheme:

where R′ and R″ are molecules of interest, and each R is independentlyan alkyl, a substituted alkyl, an aryl or a substituted aryl, and whereoptionally the R groups of the boronic ester may be cyclically linked.

In some instances, the alkynyl reagent is a DIBO, DIBAC or BARACreagent, or derivative thereof. In certain cases, the alkynyl reagent isa DIBO, DIBAC or BARAC reagent described by one of the following:

where R includes a molecule of interest (e.g., a detectable label),connected to the cyclooctyne group via an optional linker.

In some embodiments, the alkynyl reagent is described by one of thefollowing:

wherein: n is 0 to 8; p is 0 or 1;

R3 is selected from the group consisting of [(L)p-Q], hydrogen, halogen,C1-groups, C6-C24 (hetero)aryl groups, C7-C24 alkyl(hetero)aryl groupsand C7-C24 (hetero)arylalkyl groups, the alkyl groups optionally beinginterrupted by one of more hetero-atoms selected from the groupconsisting of O, N and S, wherein the alkyl groups, (hetero)aryl groups,alkyl(hetero)aryl groups and (hetero)arylalkyl groups are independentlyoptionally substituted with one or more substituents independentlyselected from the group consisting of C1-C12 alkyl groups, C2-C12alkenyl groups, C2-C12 alkynyl groups, C3-C12 cycloalkyl groups, C1-C12alkoxy groups, C2-C₁₂ alkenyloxy groups, C2-C12 alkynyloxy groups,C3-C12 cycloalkyloxy groups, halogens, amino groups, oxo groups andsilyl groups, wherein the alkyl groups, alkenyl groups, alkynyl groups,cycloalkyl groups, alkoxy groups, alkenyloxy groups, alkynyloxy groupsand cycloalkyloxy groups are optionally substituted, the alkyl groups,the alkoxy groups, the cycloalkyl groups and the cycloalkoxy groupsbeing optionally interrupted by one of more hetero-atoms selected fromthe group consisting of O, N and S, wherein the silyl groups arerepresented by the formula (R4)₃Si—, wherein R4 is independentlyselected from the group consisting of C1-C12 alkyl groups, C2-alkenylgroups, C2-C12 alkynyl groups, C3-C12 cycloalkyl groups, C1-C12 alkoxygroups, C2-C₁₋₂ alkenyloxy groups, C2-C12 alkynyloxy groups and C3-C12cycloalkyloxy groups, wherein the alkyl groups, alkenyl groups, alkynylgroups, cycloalkyl groups, alkoxy groups, alkenyloxy groups, alkynyloxygroups and cycloalkyloxy groups are optionally substituted, the alkylgroups, the alkoxy groups, the cycloalkyl groups and the cycloalkoxygroups being optionally interrupted by one of more hetero-atoms selectedfrom the group consisting of O, N and S;

L is a linking group selected from linear or branched C1-C₂₄ alkylenegroups, C24 alkenylene groups, C2-C24 alkynylene groups, C3-C24cycloalkylene groups, C5-C24 cycloalkenylene groups, C8-C24cycloalkynylene groups, C7-C24 alkyl(hetero)arylene groups, C7-C24(hetero)arylalkylene groups, C8-C24 (hetero)arylalkenylene groups,C9-C24 (hetero)arylalkynylene groups, the alkylene groups, alkenylenegroups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups,cycloalkynylene groups, alkyl(hetero)arylene groups,(hetero)arylalkylene groups, (hetero)arylalkenylene groups and(hetero)arylalkynylene groups optionally being substituted with one ormore substituents independently selected from the group consisting ofC1-C12 alkyl groups, C2-C12 alkenyl groups, C2-C12 alkynyl groups,C3-C12 cycloalkyl groups, C5-C12 cycloalkenyl groups, C8-C12cycloalkynyl groups, C1-C12 alkoxy groups, C2-C12 alkenyloxy groups,C2-C12 alkynyloxy groups, C3-C12 cycloalkyloxy groups, halogens, aminogroups, oxo and silyl groups, wherein the silyl groups can berepresented by the formula (R4)₃Si—, wherein R4 is defined as above;

Q is a molecule of interest or a functional group selected from thegroup consisting of hydrogen, halogen, R6, —CH═C(R6)₂, —C≡CR6,—[C(R6)₂O]_(q)—R6, wherein q is in the range of 1 to 200, —CN, —N3,—NCX, —XCN, —XR6, —N(R6)2, —⁺N(R6)₃, —C(X)N(R6)₂, —C(R6)₂XR6, —C(X)R6,—C(X)XR6, —S(0)R6, —S(0)₂R6, —S(0)OR6, —S(0)₂OR6, —S(0)N(R6)₂,—S(0)₂N(R6)₂, —OS(0)R6, —OS(0)₂R6, —OS(0)OR6, —OS(0)₂OR6,—P(0)(R6)(OR6), —P(0)(OR6)₂, —OP(0)(OR6)₂, —Si(R6)₃, —XC(X)R6,—XC(X)XR6, —XC(X)N(R6)₂, —N(R6)C(X)R6, —N(R6)C(X)XR6 and—N(R6)C(X)N(R6)₂, wherein X is oxygen or sulfur and wherein R6 isindependently selected from the group consisting of hydrogen, halogen,C1-C₂₄ alkyl groups, C6-C24 (hetero)aryl groups, C7-C24alkyl(hetero)aryl groups and C7-C24 (hetero)arylalkyl groups;

R1 is a molecule of interest or is independently selected from the groupconsisting of hydrogen, C1-C₂₄ alkyl groups, C6-C24 (hetero)aryl groups,C7-C24 alkyl(hetero)aryl groups and C7-C24 (hetero)arylalkyl groups; and

R2 is a molecule of interest or is independently selected from the groupconsisting of halogen, —OR6, —NO₂, —CN, —S(O)₂R6, C1-C12 alkyl groups,C1-C12 aryl groups, C1-C12 alkylaryl groups and C1-C12 arylalkyl groups,wherein R6 is as defined above, and wherein the alkyl groups, arylgroups, alkylaryl groups and arylalkyl groups are optionallysubstituted.

Phosphine Reagents

In some cases, the azide-reactive reagent includes a phosphinefunctional group. Without wishing to be bound by theory, reaction of theazido group with a phosphine reagent may lead to an iminophosphorane(aza-ylide) intermediate, which can react intramolecularly with anadjacent electrophilic group to produce a covalent amide bond.

Azido-reactive reagents of interest include, but are not limited to,dithiols and phosphines such as, arylphosphines (e.g., a triphenylphosphine), alkylphosphines (e.g., a trialkylphosphine such astris(2-carboxyethyl)phosphine (TCEP)), or arylalkylphosphines.

Reagents Reactive with Alkyne-Modified Amino Acids

Any convenient reagents reactive with alkyne-modified D-amino acids maybe utilized in the subject reagents and methods. Alkyne-reactivereagents of interest include, but are not limited to, click chemistry orcopper-free click chemistry reagents, and azide-containing reagents.

In some instances, the azide-containing reagent is described by theformula:

N₃-L-Y

wherein: L is a linker; and Y is a molecule of interest, e.g., adetectable label.

In some embodiments, L is (T)_(n), where each n is a number selectedfrom zero to 40; and where each T is a divalent moiety independentlyselected from alkylene, substituted alkylene, alkenylene, substitutedalkenylene, alkynylene, substituted alkynylene, arylene, substitutedarylene, cycloalkylene, substituted cycloalkylene, heteroarylene,substituted heteroarylene, heterocyclene, substituted heterocyclene,acyl, amido, acyloxy, urethanylene, thioester, sulfonyl, sulfonamide,sulfonyl ester, —O—, —S—, —NH—, and substituted amine.

Exemplary azide-containing groups that may be adapted for use in thesubject reagents include, but are not limited to: azidoacetylmannosamine(ManNAz), azido-derivatized sugar (e.g., fucose),5′-diphospho-6-azido-B-L-fucopyranoside, 3-azido-7-hydroxycoumarin,4-azido-N-ethyl-1,8-naphthalimide, azide-linked biotin, azide-labelledantibody, an azide containing amino acid or peptide (e.g., azidogylcineor azidoalanine), etc.

A subject azide-containing reagent may be prepared using any convenientmethod, including but not limited to conversion of the amino group of anamine-containing reagent into an azide using any suitable method andchemistry. Methods to introduce an azide group are for example disclosedin WO03/003806.

Reagents Reactive with Norbornene-Modified Amino Acids

Any convenient reagents reactive with norbornene-modified D-amino acidsmay utilized in the subject reagents and methods. Norbornene-reactivereagents of interest include, but are not limited to, hydrazonoylchloride-containing reagents, photo-click reagents, tetrazole-containingreagents, tetrazine-containing reagents, reagents such as thosedescribed by Carell et al. “A Genetically Encoded Norbornene Amino Acidfor the Mild and Selective Modification of Proteins in a Copper-FreeClick Reaction”, Angew. Chem. Int. Ed. 1012, 51, 4466-4469.

In some instances, the norbornene-reactive reagent is described by theformula:

W-L-Y

wherein: L is a linker; W is a norbornene reactive functional group; andY is a molecule of interest, e.g., a detectable label.

In some embodiments, L is (T)_(n), where each n is a number selectedfrom zero to 40; and where each T is a divalent moiety independentlyselected from alkylene, substituted alkylene, alkenylene, substitutedalkenylene, alkynylene, substituted alkynylene, arylene, substitutedarylene, cycloalkylene, substituted cycloalkylene, heteroarylene,substituted heteroarylene, heterocyclene, substituted heterocyclene,acyl, amido, acyloxy, urethanylene, thioester, sulfonyl, sulfonamide,sulfonyl ester, —O—, —S—, —NH—, and substituted amine.

In some instances, W is selected from a tetrazine, a tetrazole, ahydrazonyl chloride or a nitrile imine. In some cases, W is a functionalgroup that is capable of reacting directly with a norbornene-modifiedD-amino acid. In other cases, W may first be activated by contact with astimulus, such as a light stimulus or a chemical catalyst.

In some embodiments, the norbornene-reactive reagent is described by oneof the following structures:

Modified Peptidoglycan

In some embodiments, a modified PG is described by one of the followingstructures: PG-L-N₃ or PG-L-X

where PG is a peptidoglycan, L is an optional linker, X is an alkynegroup, a norbornene group or a tetrazole group (e.g., as describedherein), and —N₃ is an azido group. In some instances, PG includes aD-amino acid residue. In certain instances, the azido or the alkyne ornorbornene group X is connected to the D-amino acid residue of the PGvia the optional linker L. As such, the D-amino acid residue may bereferred to as a modified D-amino acid residue. The modified D-aminoacid residue may be included at any convenient position of the PGsequence.

The modified PGs may be conjugated to a reagent, e.g., as describedherein, convenient chemistry. Conjugation chemistries of interestinclude, but are not limited to, click chemistry, photo-click chemistry,tetrazine-click chemistry, and copper-free click chemistry, e.g.,conjugations that include the [3+2] cycloaddition reaction of azide andalkyne functional groups to produce a 1,2,3-triazole linkage, or areversed-electron-demand Diers-Alder reaction of a tetrazine and anorbornene functional group, or a photoinduced 1,3-dipolar cycloadditionreaction of a tetrazole and an alkene. In some instances, the modifiedPG includes an alkynyl group and the reagent includes an azido group. Inother instances, the modified PG includes an azido group and the reagentincludes an alkynyl group. In certain instances, the modified PGincludes a norbornene group and the reagent includes a tetrazine or atetrazole group. In certain cases, the modified PG includes a tetrazolegroup and the reagent includes an alkene. As such, in certainembodiments, a modified PG of the present disclosure may be labeled toinclude a PG linked to a molecule of interest via aheterocycle-containing linker, such as a triazole linker, a linkercontaining the product of a reversed-electron-demand Diers-Alderreaction, e.g., a norborene-tetrazine derived heterocycle containinglinker as described herein, or a pyrazoline-containing linker.

In some instances, the labeled PG is described by the following formula:

PG-L¹-Z-L²-Y

where PG is a modified peptidoglycan; Y is a molecule of interest (e.g.,a detectable label); L¹ and L² are independently optional linkers; and Zis a 1,2,3-triazole. The labeled PG may be derived from the conjugationof any convenient modified PG (e.g., as described herein) and anyconvenient alkynyl reagent or an azide-containing reagent (e.g., asdescribed herein). In some cases, Y is a detectable label.

In certain cases, Z is a 1,2,3-triazole, a pyrazoline or thecycloaddition product of a norbornene and a tetrazine, a tetrazole or ahydrazonoyl chloride. In certain instances, Z is a norborene-tetrazinederived heterocycle described by the following structure:

In certain embodiments, the labeled PG includes a detectable label(e.g., a florescent label).

Detectable Labels

Exemplary detectable labels include, but are not necessarily limited to,molecules (e.g., autofluorescent molecules, molecules that fluoresceupon contact with a reagent, etc.), radioactive labels (e.g., ¹¹¹In,¹²⁵I, ¹³¹I, ²¹²B, ⁹⁰Y, ¹⁸⁶Rh, and the like); biotin (e.g., to bedetected through reaction of biotin and avidin); fluorescent tags;imaging reagents (e.g., those described in U.S. Pat. No. 4,741,900 andU.S. Pat. No. 5,326,856), and the like. Detectable labels also includepeptides or polypeptides that can be detected by antibody binding, e.g.,by binding of a detectably labeled antibody or by detection of boundantibody through a sandwich-type assay. Also suitable for use arequantum dots (e.g., detectably labeled semiconductor nanocrystals, suchas fluorescently labeled quantum dots, antibody-conjugated quantum dots,and the like). See, e.g., Dubertret et al. 2002 Science 298:759-1762;Chan et al. (1998) Science 281:2016-2018; U.S. Pat. No. 6,855,551;Bruchez et al. (1998) Science 281:2013-2016

Suitable fluorescent molecules (fluorophores) include, but are notlimited to, fluorescein isothiocyanate, succinimidyl esters ofcarboxyfluorescein, succinimidyl esters of fluorescein, 5-isomer offluorescein dichlorotriazine, cagedcarboxyfluorescein-alanine-carboxamide, Oregon Green 488, Oregon Green514; Lucifer Yellow, acridine Orange, rhodamine, tetramethylrhodamine,Texas Red, propidium iodide, JC-1(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazoylcarbocyanineiodide), tetrabromorhodamine 123, rhodamine 6G, TMRM(tetramethylrhodamine-, methyl ester), TMRE (tetramethylrhodamine, ethylester), tetramethylrosamine, rhodamine B and4-dimethylaminotetramethylrosamine, green fluorescent protein,blue-shifted green fluorescent protein, cyan-shifted green fluorescentprotein, red-shifted green fluorescent protein, yellow-shifted greenfluorescent protein,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives: acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphth-alimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide;4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a diaza-5-indacene-3-propioni-cacid BODIPY; cascade blue; Brilliant Yellow; coumarin and derivatives:coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin120),7-amino-4-trifluoromethylcoumarin (Coumarin 151); cyanine dyes;cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriaamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2-,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-(dimethylamino)naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives: eosin, eosin isothiocyanate, erythrosin and derivatives:erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives: 5-carboxyfluorescein(FAM),5-(4,6-dichlorotriazin-2-yl)amino-1-fluorescein (DTAF),2′,7′dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelli-feroneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl hodamine isothiocyanate (TRITC); riboflavin;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid(EDANS),4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), rosolicacid; CAL Fluor Orange 560; terbium chelate derivatives; Cy 3; Cy 5; Cy5.5; Cy 7; IRD 700; IRD 800; La Jolla Blue; phthalo cyanine; andnaphthalo cyanine, coumarins and related dyes, xanthene dyes such asrhodols, resorufins, bimanes, acridines, isoindoles, dansyl dyes,aminophthalic hydrazides such as luminol, and isoluminol derivatives,aminophthalimides, aminonaphthalimides, aminobenzofurans,aminoquinolines, dicyanohydroquinones, and fluorescent europium andterbium complexes; and the like. Fluorophores of interest are furtherdescribed in WO 01/42505 and WO 01/86001.

Suitable fluorescent proteins and chromogenic proteins include, but arenot limited to, a green fluorescent protein (GFP), including, but notlimited to, a GFP derived from Aequoria victoria or a derivativethereof, e.g., a “humanized” derivative such as Enhanced GFP, which isavailable commercially, e.g., from Clontech, Inc.; a GFP from anotherspecies such as Renilla reniformis, Renilla mulleri, or Ptilosarcusguemyi, as described in, e.g., WO 99/49019 and Peelle et al. (2001) J.Protein Chem. 20:507-519; “humanized” recombinant GFP (hrGFP)(Stratagene); any of a variety of fluorescent and colored proteins fromAnthozoan species, as described in, e.g., Matz et al. (1999) NatureBiotechnol. 17:969-973; and the like.

Modified Bacteria

The present disclosure provides modified bacteria having incorporatedinto the PG of the bacteria modified PG (PG comprising a modifiedD-amino acid that includes a bioorthogonal functional group such as anazide, an alkyne, or a norbornene group), as described above.

Bacteria that can be modified such that PG present in the bacterial cellwall being synthesized comprises at least one modified D-amino acidinclude, but are not limited to, pathogenic intracellular bacteria(i.e., pathogenic bacteria that replicate in a host cell); pathogenicbacteria that do not replicate in a host cell; and non-pathogenicbacteria (e.g., non-pathogenic laboratory strains of bacteria).Non-pathogenic bacteria include commensal bacteria (present in a host)as well as free-living bacteria (living outside a host).

In some cases, the bacterium is an obligate intracellular pathogen or afacultative intracellular pathogen. Examples of such bacteria include,e.g., a Mycobacterium species. Examples of species of Mycobacteriuminclude, but are not limited to, M. tuberculosis, M. bovis, M. bovisstrain Bacillus calmette-guerin (BCG) including BCG substrains, M.avium, M. intracellulare, M. africanunum, M. kansasii, M. marinum, M.ulcerans and M. paratuberculosis. Examples of other obligate andfacultative intracellular bacterial species include, but are not limitedto, Legionella pneumophila, other Legionella species, Salmonella typhi,other Salmonella species, Shigella species, Listeria monocytogenes,Staphylococcus aureus, Staphylococcus epidermidis, Bacteroides fragilis,other Bacteroides species, Chlamydia pneumoniae, Chlamydia trachomatis,Chlamydia psittaci, Coxiella burnetii, other Rickettsial species, andEhrlichia species.

Suitable bacteria include, but are not limited to, Francisellatularensis; Listeria monocytogenes; Salmonella; Brucella; Legionellapneumophila; Mycobacterium (e.g., M. tuberculosis, M. leprae, M. bovis,M. avium, M. abscessus); Nocardia (e.g., N. asteroids, N. farcinica, N.nova, N. transvalensis, N. brasiliensis, N. pseudobrasiliensis);Rhodococcus equui; Yersinia pestis; Neisseria (e.g., N. meningitidis, N.gonorrhoeae); Shigella (e.g., S. dysenteriae, S. flexneri, S. boydii,and S. sonnei); Chlamydia (C. trachomatis, C. pneumoniae, C. psittaci);Rickettsia; and Coxsiella. Other suitable bacteria include, e.g.,pathogens such as Vibrio cholerae, Pseudomonas aeruginosa, andpathogenic Escherichia coli; model organisms such as Escherichia coli,Bacillus subtilis, and Caulobacter cresentus; facultative pathogens suchas Streptococcal and Clostridial species; and commensals such asBacteroides thetaiotamicron.

Methods of Identifying Anti-Microbial Agents

The present disclosure provides methods of identifying anti-microbialagents, the methods generally involving: a) contacting a bacterial cellwith a test agent; and b) determining the effect, if any, of the testagent on incorporation of a modified D-amino acid (a modified D-aminoacid that includes a bioorthogonal functional group such as an azide, analkyne, or a norbornene group, as described above) into peptidoglycanpresent in the bacterial cell. For the discussion below, a “modifiedD-amino acid” refers to a modified D-amino acid that includes abioorthogonal functional group such as an azide, an alkyne, or anorbornene group, as described above.

Whether a test agent reduces incorporation of a-modified D-amino acidinto PG present in a bacterial cell can be determined by contacting thebacterium with a reagent, as described above, which reacts with amodified D-amino acid present in the PG, where the reagent comprises adetectable label. In some cases, the detectable label is a fluorescentlabel. Where a test agent inhibits incorporation of a modified D-aminoacid into PG present in a bacterial cell, the amount of label in the PGis reduced.

Test agents of interest include test agents that inhibit incorporationof a modified D-amino acid into the PG of a bacterial cell by at leastabout 10%, at least about 20%, at least about 25%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, or more than 80%, compared to the extentof incorporation of the modified D-amino acid into the PG in the absenceof the test agent. Test agents that inhibit incorporation of a modifiedD-amino acid into the PG of a bacterial cell are considered candidateanti-microbial agents.

In some embodiments, the bacterium is present in a eukaryotic cell-freeliquid medium, i.e., the bacterium is not present intracellularly in aeukaryotic host cell. In other cases, the bacterium is presentintracellularly in a eukaryotic host cell.

As noted above, in some instances, the bacterium is presentintracellularly in a eukaryotic host cell. Suitable eukaryotic hostcells include, but are not limited to, macrophages, monocytes, dendriticcells, and the like. Suitable cells include, e.g., eukaryotic cells,e.g., mammalian cells such as primary cells (e.g., bone marrow-derivedmacrophages; peripheral blood mononuclear cells; etc.). Suitable cellsinclude, e.g., eukaryotic cells, e.g., mammalian cells such as humanumbilical vein endothelial cells (HUVEC; e.g., American Type CultureCollection (ATCC)CRL-1730), human microvascular endothelial cells(HMEC-1; ATCC CRL-4025), PC3 cells (ATCC CRL1435), MDA-MB-231 cells(ATCC HTB26), MCF-7 cells (ATCC HTB22), HeLa cells (ATCC No. CCL-2), CHOcells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCCNo. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658),Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No.CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse Lcells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No.CRL1573), and the like. In one non-limiting example, the cell used is aJ774 macrophage cell line. In one non-limiting example, the cell used isthe J774A.1 macrophage cell line (American Type Culture Collection(ATCC) TIB-67).

Suitable bacteria include, but are not limited to, facultativeintracellular bacteria and obligate intracellular bacteria. Suitablebacteria include, but are not limited to, Francisella tularensis;Listeria monocytogenes; Salmonella; Brucella; Legionella pneumophila;Mycobacterium (e.g., M. tuberculosis, M. leprae, M. bovis, M. avium, M.abscessus); Nocardia (e.g., N. asteroids, N. farcinica, N. nova, N.transvalensis, N. brasiliensis, N. pseudobrasiliensis); Rhodococcusequui; Yersinia pestis; Neisseria (e.g., N. meningitidis, N.gonorrhoeae); Shigella (e.g., S. dysenteriae, S. flexneri, S. boydii,and S. sonnei); Chlamydia (C. trachomatis, C. pneumoniae, C. psittaci);Rickettsia; and Coxsiella. Other suitable bacteria include, e.g.,pathogens such as Vibrio cholerae, Pseudomonas aeruginosa, andpathogenic Escherichia coli; model organisms such as Escherichia coli,Bacillus subtilis, and Caulobacter cresentus; facultative pathogens suchas Streptococcal and Clostridial species; and commensals such asBacteroides thetaiotamicron.

As used herein, the term “determining” refers to both quantitative andqualitative determinations and as such, the term “determining” is usedinterchangeably herein with “assaying,” “measuring,” and the like.

The terms “candidate agent,” “test agent,” “agent,” “substance,” and“compound” are used interchangeably herein. Candidate agents encompassnumerous chemical classes, typically synthetic, semi-synthetic, ornaturally-occurring inorganic or organic molecules. Candidate agentsinclude those found in large libraries of synthetic or naturalcompounds. For example, synthetic compound libraries are commerciallyavailable from Maybridge Chemical Co. (Trevillet, Cornwall, UK),ComGenex (South San Francisco, Calif.), and MicroSource (New Milford,Conn.). A rare chemical library is available from Aldrich (Milwaukee,Wis.). Alternatively, libraries of natural compounds in the form ofbacterial, fungal, plant and animal extracts are available from Pan Labs(Bothell, Wash.) or are readily producible.

Candidate agents may be small organic or inorganic compounds having amolecular weight of more than 50 and less than about 10,000 daltons,e.g., a candidate agent may have a molecular weight of from about 50daltons to about 100 daltons, from about 100 daltons to about 150daltons, from about 150 daltons to about 200 daltons, from about 200daltons to about 500 daltons, from about 500 daltons to about 1000daltons, from about 1,000 daltons to about 2500 daltons, from about 2500daltons to about 5000 daltons, from about 5000 daltons to about 7500daltons, or from about 7500 daltons to about 10,000 daltons. Candidateagents may comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and mayinclude at least an amine, carbonyl, hydroxyl or carboxyl group, and maycontain at least two of the functional chemical groups. The candidateagents may comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

Assays of the invention include controls, where suitable controlsinclude a sample without the test agent (e.g., a sample the bacterialcell in the absence of the test agent). Generally a plurality of assaymixtures is run in parallel with different agent concentrations toobtain a differential response to the various concentrations. Typically,one of these concentrations serves as a negative control, i.e. at zeroconcentration or below the level of detection.

A variety of other reagents may be included in the screening assay.These include reagents such as salts, neutral proteins, e.g. albumin,detergents, etc., including agents that are used to facilitate optimalenzyme activity and/or reduce non-specific or background activity.Reagents that improve the efficiency of the assay, such as proteaseinhibitors, etc. may be used. The components of the assay mixture areadded in any order that provides for the requisite activity. Incubationsare performed at any suitable temperature, typically between 4° C. and40° C. Incubation periods are selected for optimum activity, but mayalso be optimized to facilitate rapid high-throughput screening.Typically between 0.1 hour and 1 hour will be sufficient.

Immunomodulatory Compounds and Compositions

The present disclosure provides immunomodulatory compounds, andcompositions comprising such compounds. A subject immunomodulatorycompound comprises a PG fragment comprising a modified D-amino acid (amodified D-amino acid that includes a bioorthogonal functional groupsuch as an azide, an alkyne, or a norbornene group, as described above)conjugated to a second compound (e.g., a conjugate partner). Suitableconjugate partners include, e.g., a therapeutic agent; an antigen; anallergen; a non-PG immunomodulatory agent; and the like. A subjectimmunomodulatory compound is useful for modulating (e.g., enhancing animmune response; reducing an immune response; shifting an immuneresponse (e.g., shifting from a Th1 lymphocyte response to a Th2lymphocyte response; shifting toward a Th17 lymphocyte response; etc.))in an individual. In the discussion below, a “modified D-amino acid”refers to a modified D-amino acid that includes a bioorthogonalfunctional group such as an azide, an alkyne, or a norbornene group, asdescribed above.

Suitable therapeutic agents include, but are not limited to, ananti-viral agent, an anti-bacterial agent, an anti-fungal agent, acytokine, a hormone, an antibody, etc.

Suitable therapeutic agents include, but are not limited to,azathioprine, cyclosporin methothrexate, leflunomide, corticosteroids,cyclophosphamide, cyclosporine A, cyclosporin G, mycophenolate mofetil,ascomycin, rapamycin (sirolimus), FK-506, mizoribine, deoxyspergualin,brequinar, mycophenolic acid, malononitriloamindes (e.g., leflunamide),T cell receptor modulators, and cytokine receptor modulators, peptidemimetics, and antibodies (e.g., human, humanized, chimeric, monoclonal,polyclonal, Fvs, ccFvs, Fab, single domain antibodies (nanobodies), orF(ab)₂ fragments or epitope binding fragments), nucleic acid molecules(e.g., antisense nucleic acid molecules, siRNA, and triple helices),small molecules, organic compounds, and inorganic compounds. Examples ofT cell receptor modulators include, but are not limited to, anti-T cellreceptor antibodies (e.g., anti-CD4 antibodies (e.g., cM-T412(Boehringer), IDEC-CE9.1™ (IDEC and SKB), mAB 4162W94, Orthoclone andOKTcdr4a (Janssen-Cilag)), anti-CD3 antibodies (such as, by way ofexample only, Nuvion (Product Design Labs), OKT3 (Johnson & Johnson), orRituxan (IDEC)), anti-CD5 antibodies (e.g., an anti-CD5 ricin-linkedimmunoconjugate), anti-CD7 antibodies (e.g., CHH-380 (Novartis)),anti-CD8 antibodies, anti-CD40 ligand monoclonal antibodies (e.g.,IDEC-131 (IDEC)), anti-CD52 antibodies (e.g., CAMPATH 1H (Ilex)),anti-CD2 antibodies, anti-CD11a antibodies (e.g., Xanelim (Genentech)),anti-B7 antibodies (e.g., IDEC-114 (IDEC)), CTLA4-immunoglobulin, andother toll receptor-like (TLR) modulators. Examples of cytokine receptormodulators include, but are not limited to, soluble cytokine receptors(e.g., the extracellular domain of a TNF-α receptor or a fragmentthereof, the extracellular domain of an IL-1β receptor or a fragmentthereof, and the extracellular domain of an IL-6 receptor or a fragmentthereof), cytokines or fragments thereof (e.g., interleukin (IL)-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15,TNF-α, interferon (IFN)-α, IFN-β, IFN-γ, and GM-CSF), anti-cytokinereceptor antibodies (e.g., anti-IFN receptor antibodies, anti-IL-2receptor antibodies (e.g., Zenapax (Protein Design Labs)), anti-IL-4receptor antibodies, anti-IL-6 receptor antibodies, anti-IL-10 receptorantibodies, and anti-IL-12 receptor antibodies), anti-cytokineantibodies (e.g., anti-IFN antibodies, anti-TNF-α antibodies, anti-IL-1βantibodies, anti-IL-6 antibodies, anti-IL-8 antibodies (e.g., ABX-IL-8(Abgenix)), and anti-IL-12 antibodies).

Suitable cytokines and modulators of cytokine function include, but arenot limited to, interleukin-2 (IL-2), interleukin-3 (IL-3),interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6),interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-10 (IL-10),interleukin-12 (IL-12), interleukin 15 (IL-15), interleukin 18 (IL-18),platelet derived growth factor (PDGF), erythropoietin (Epo), epidermalgrowth factor (EGF), fibroblast growth factor (FGF), granulocytemacrophage stimulating factor (GM-CSF), granulocyte colony stimulatingfactor (G-CSF), macrophage colony stimulating factor (M-CSF), prolactin,alpha-, beta-, and gamma-interferon, interferon β-1a, interferon β-1b,interferon α-1, interferon α-2a (roferon), interferon α-2b, pegylatedinterferons (by way of example only, peginterferon α-2a andpeginterferon α-2b), intron, Peg-Intron, Pegasys, consensus interferon(infergen), albumin-interferon α and albuferon.

Suitable antigens include, antigens derived from infectious agents(e.g., bacterial, fungal (including unicellular and multicellular), andviral infectious agents); tumor antigens; and the like. Examples ofpathogens include, but are not limited to, viruses, e.g., herpes simplexvirus (HSV), hepatitis A virus, hepatitis B virus (HBV), hepatitis Cvirus (HCV), cytomegalovirus (CMV), dengue virus, flavivirus,Epstein-Barr virus (EBV), influenza virus, measles virus, humanimmunodeficiency virus (HIV), human papilloma virus (HPV), Japaneseencephalitis virus, norovirus, polio virus, rotavirus, respiratorysyncytial virus (RSV), ebola virus, rabies virus, Sendai virus, severeacute respiratory syndrome (SARS) coronavirus, smallpox virus, West Nilevirus, yellow fever virus; bacteria, e.g., Mycobacterium tuberculosis(tuberculosis), Chlamydia trachomatis (trachoma), Haemophilus influenzae(otitis media), Neisseria meningitidis (meningitis), Streptococcuspneumoniae (pneumonia), Escherichia coli (intestimal disorders)Staphylococcus aureus, Bacillus anthracis (anthrax), Borreliaburgdorferi (Lyme's disease); and parasites, e.g., Plasmodium (malaria),Leishmania, Trypanosoma cruzi, Trypanosoma brucei, Ascaris lumbricoides(ascariasis), hookworm, Onchocerca volvulus (river blindness),Schistosoma (schistosomasis), Trichuris trichiura (trichurasis).

Suitable allergens include, but are not limited to, allergens such asreactive major dust mite allergens Der pI and Der pll; T cell epitopepeptides of the Der pII allergen; Amb al ragweed pollen allergen;phospholipase A2 (bee venom) allergen and T cell epitopes; white birchpollen (Betyl); the Fel dl major domestic cat allergen; tree pollen;grass pollen; inhaled allergens (e.g., grass, weed, and tree pollens,mold spores, chemicals, cockroach calyx, dust mite excretions, animaldander, saliva); ingested allergens (e.g., food, food supplements, homeremedies, medications); contact allergens (e.g., cosmetics, fragrances,plants, detergents, chemicals, metals, latex); and injected allergens(e.g., medications, insect venom).

A subject immunomodulatory compound can be administered orally asdiscrete dosage forms, wherein such dosage forms include, but are notlimited to, capsules, gelatin capsules, caplets, tablets, chewabletablets, powders, granules, syrups, flavored syrups, solutions orsuspensions in aqueous or non-aqueous liquids, edible foams or whips,and oil-in-water liquid emulsions or water-in-oil liquid emulsions.

The capsules, gelatin capsules, caplets, tablets, chewable tablets,powders or granules, used for the oral administration of a subjectimmunomodulatory compound are prepared by admixing a subjectimmunomodulatory compound (active ingredient) together with at least oneexcipient using conventional pharmaceutical compounding techniques.Non-limiting examples of excipients used in oral dosage forms describedherein include, but are not limited to, binders, fillers, disintegrants,lubricants, absorbents, colorants, flavors, preservatives andsweeteners.

Non-limiting examples of suitable binders include, but are not limitedto, corn starch, potato starch, starch paste, pre-gelatinized starch, orother starches, sugars, gelatin, natural and synthetic gums such asacacia, sodium alginate, alginic acid, other alginates, tragacanth, guargum, cellulose and its derivatives (by way of example only, ethylcellulose, cellulose acetate, carboxymethyl cellulose calcium, sodiumcarboxymethylcellulose, methyl cellulose, hydroxypropyl methylcelluloseand microcrystalline cellulose), magnesium aluminum silicate, polyvinylpyrrolidone and combinations thereof.

Non-limiting examples of suitable fillers include, but are not limitedto, talc, calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof. Incertain embodiments, the binder or filler in pharmaceutical compositionsprovided herein are present in from about 50 to about 99 weight percentof the pharmaceutical composition or dosage form.

Non-limiting examples of suitable disintegrants include, but are notlimited to, agar-agar, alginic acid, sodium alginate, calcium carbonate,sodium carbonate, microcrystalline cellulose, croscarmellose sodium,crospovidone, polacrilin potassium, sodium starch glycolate, potato ortapioca starch, pre-gelatinized starch, other starches, clays, otheralgins, other celluloses, gums, and combinations thereof. In certainembodiments, the amount of disintegrant used in the pharmaceuticalcompositions provided herein is from about 0.5 to about 15 weightpercent of disintegrant, while in other embodiments the amount is fromabout 1 to about 5 weight percent of disintegrant.

Non-limiting examples of suitable lubricants include, but are notlimited to, stearate, calcium stearate, magnesium stearate, stearicacid, mineral oil, light mineral oil, glycerin, sorbitol, mannitol,polyethylene glycol, other glycols, sodium lauryl sulfate, talc,hydrogenated vegetable oil (by way of example only, peanut oil,cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, sodium oleate, ethyl oleate, ethyllaureate, agar, silica, a syloid silica gel (AEROSIL 200, manufacturedby W.R. Grace Co. of Baltimore, Md.), a coagulated aerosol of syntheticsilica (marketed by Degussa Co. of Plano, Tex.), CAB-O-SIL (a pyrogenicsilicon dioxide product sold by Cabot Co. of Boston, Mass.) andcombinations thereof. In certain embodiments, the amount of lubricantsused in the pharmaceutical compositions provided herein is in an amountof less than about 1 weight percent of the pharmaceutical compositionsor dosage forms.

Non-limiting examples of suitable diluents include, but are not limitedto, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycineor combinations thereof.

In certain embodiments, a subject immunomodulatory compound is orallyadministered as a controlled release dosage form. Such dosage forms areused to provide slow or controlled-release of a subject immunomodulatorycompound. Controlled release is obtained using, for example,hydroxypropylmethyl cellulose, other polymer matrices, gels, permeablemembranes, osmotic systems, multilayer coatings, microparticles,liposomes, microspheres, or a combination thereof.

Administration of a subject immunomodulatory compound as oral fluidssuch as solution, syrups and elixirs are prepared in unit dosage formssuch that a given quantity of solution, syrups or elixirs contains apredetermined amount of a subject immunomodulatory compound. Syrups areprepared by dissolving the compound in a suitably flavored aqueoussolution, while elixirs are prepared through the use of a non-toxicalcoholic vehicle. Suspensions are formulated by dispersing the compoundin a non-toxic vehicle. Non-limiting examples of excipients used in asoral fluids for oral administration include, but are not limited to,solubilizers, emulsifiers, flavoring agents, preservatives, and coloringagents. Non-limiting examples of solubilizers and emulsifiers include,but are not limited to, water, glycols, oils, alcohols, ethoxylatedisostearyl alcohols and polyoxy ethylene sorbitol ethers. Non-limitingexamples of preservatives include, but are not limited to, sodiumbenzoate. Non-limiting examples of flavoring agents include, but are notlimited to, peppermint oil or natural sweeteners or saccharin or otherartificial sweeteners.

In certain embodiments, pharmaceutical compositions comprising a subjectimmunomodulatory compound are administered parenterally by variousroutes including, but not limited to, subcutaneous, intravenous(including bolus injection), intramuscular, and intraarterial.

Parenteral dosage forms are administered in the form of sterile orsterilizable solutions, suspensions, dry and/or lyophylized productsready to be dissolved or suspended in a pharmaceutically acceptablevehicle for injection (reconstitutable powders) and emulsions. Vehiclesused in such dosage forms include, but are not limited to, Water forInjection USP; aqueous vehicles such as, but not limited to, SodiumChloride Injection, Ringer's Injection, Dextrose Injection, Dextrose andSodium Chloride Injection, and Lactated Ringer's Injection;water-miscible vehicles such as, but not limited to, ethyl alcohol,polyethylene glycol, and polypropylene glycol; and non-aqueous vehiclessuch as, but not limited to, corn oil, cottonseed oil, peanut oil,sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

In certain embodiments, pharmaceutical compositions comprising a subjectimmunomodulatory compound are administered transdermally. Suchtransdermal dosage forms include “reservoir type” or “matrix type”patches, which are applied to the skin and worn for a specific period oftime to permit the penetration of a desired amount of a subjectimmunomodulatory compound. By way of example only, such transdermaldevices are in the form of a bandage comprising a backing member, areservoir containing the compound optionally with carriers, optionally arate controlling barrier to deliver the compound to the skin of the hostat a controlled and predetermined rate over a prolonged period of time,and means to secure the device to the skin. In other embodiments, matrixtransdermal formulations are used.

Formulations for transdermal delivery of a subject immunomodulatorycompound include an effective amount of a subject immunomodulatorycompound, a carrier and an optional diluent. A carrier includes, but isnot limited to, absorbable pharmacologically acceptable solvents toassist passage through the skin of the host, such as water, acetone,ethanol, ethylene glycol, propylene glycol, butane-1,3-diol, isopropylmyristate, isopropyl palmitate, mineral oil, and combinations thereof.

In certain embodiments, such transdermal delivery systems includepenetration enhancers to assist in delivering a subject immunomodulatorycompound to the tissue. Such penetration enhancers include, but are notlimited to, acetone; various alcohols such as ethanol, oleyl, andtetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide; dimethylacetamide; dimethyl formamide; polyethylene glycol; pyrrolidones such aspolyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; andvarious water-soluble or insoluble sugar esters such as Tween 80(polysorbate 80) and Span 60 (sorbitan monostearate).

In other embodiments, the pH of such a transdermal pharmaceuticalcomposition or dosage form, or of the tissue to which the pharmaceuticalcomposition or dosage form is applied, is adjusted to improve deliveryof a subject immunomodulatory compound. In other embodiments, thepolarity of a solvent carrier, its ionic strength, or tonicity isadjusted to improve delivery. In other embodiments, compounds such asstearates are added to advantageously alter the hydrophilicity orlipophilicity of a subject immunomodulatory compound so as to improvedelivery. In certain embodiments, such stearates serve as a lipidvehicle for the formulation, as an emulsifying agent or surfactant, andas a delivery-enhancing or penetration-enhancing agent. In otherembodiments, different salts, hydrates or solvates of a subjectimmunomodulatory compound are used to further adjust the properties ofthe resulting composition.

In certain embodiments, pharmaceutical compositions comprising a subjectimmunomodulatory compound are administered nasally. The dosage forms fornasal administration are formulated as aerosols, solutions, drops, gelsor dry powders. In certain embodiments pharmaceutical compositionscomprising a subject immunomodulatory compound are administered rectallyin the form of suppositories, enemas, ointment, creams rectal foams orrectal gels. In certain embodiments such suppositories are prepared fromfatty emulsions or suspensions, cocoa butter or other glycerides.

Methods of Modulating an Immune Response

The present disclosure provides methods of modulating an immune responsein an individual, the methods generally involving administering to anindividual in need thereof an effective amount of an immunomodulatorycompound or composition, as described above.

An “effective amount” of a subject immunomodulatory compound orcomposition is an amount that, when administered to an individual in oneor more doses, is effective to modulate an immune response in theindividual. For example, in some case, an “effective amount” of asubject immunomodulatory compound or composition is an amount that, whenadministered to an individual in one or more doses, is effective toincrease the number of T lymphocytes (e.g., T helper cells, cytotoxic Tcells, or any T cell subset) in the individual by at least about 10%, atleast about 25%, at least about 50%, at least about 75%, at least about2-fold, at least about 5-fold, at least about 10-fold, or more than10-fold, compared to the number of T lymphocytes in the individualbefore administration of the subject immunomodulatory compound orcomposition.

As another example, in some cases, an “effective amount” of a subjectimmunomodulatory compound or composition is an amount that, whenadministered to an individual in one or more doses, is effective toincrease the number of B cells in the individual by at least about 10%,at least about 25%, at least about 50%, at least about 75%, at leastabout 2-fold, at least about 5-fold, at least about 10-fold, or morethan 10-fold, compared to the number of B cells in the individual beforeadministration of the subject immunomodulatory compound or composition.

As another example, in some cases, an “effective amount” of a subjectimmunomodulatory compound or composition is an amount that, whenadministered to an individual in one or more doses, is effective toincrease the amount of circulating immunoglobulin in the individual byat least about 10%, at least about 25%, at least about 50%, at leastabout 75%, at least about 2-fold, at least about 5-fold, at least about10-fold, or more than 10-fold, compared to the level of circulatingimmunoglobulin in the individual before administration of the subjectimmunomodulatory compound or composition.

Suitable routes of administration include, e.g., parenteral and enteralroutes of administration. Suitable routes of administration include, butare not limited to, intranasal, oral, mucosal, sublingual, transdermal,and transmucosal. A subject immunomodulatory compound or composition canbe administered via oral administration, rectal administration,parenteral, intravenous administration, intravitreal administration,intramuscular administration, inhalation, intranasal administration,topical administration, ophthalmic administration, or oticadministration.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1 Preparation of Cyclooctyne-Functionalized D-Amino Acids

Cyclooctyne-functionalized D-amino acids are prepared fromFmoc-protected D-diaminopropionic acid, which are prepared in one stepfrom commercially available Fmoc-D-asparagine using a procedure asdescribed by Lau et al. (Synlett 2011, 13, 1917-1919) (Scheme 1). TheFmoc-protected D-diaminopropionic acid was then coupled tocyclooctynol-para-nitrophenol carbonates 24 to 26 to generateFmoc-protected cyclooctyne D-amino acids 27 to 29 using methods asdescribed by Plass et al. (Angew. Chem. Int. Ed. 2011, 50, 3878-3881)and Dommerholt et al. (Angew. Chem. Int. Ed. 2010, 49, 9422-9425).Finally, these compounds were deprotected using piperazine resin inCH₂C₁₂ to yield the cyclooctyne amino acids 30 (octDala), 31(exobcnDala), and 32 (endobcnDala).

With all three amino acids in hand, their incorporation into thepeptidoglycan of gram-positive bacteria was tested. The bacteria weregrown for one-doubling time in the presence of 5 mM unnatural aminoacids 30 to 32. The cells were then washed to remove excess amino acidand labeled with 20 μM of the commercially availableazido-PEG3-carboxyrhodamine. This fluorophore efficiently labelsterminal-alkyne-tagged peptidoglycan under copper-click conditions. Thecells were then washed to remove excess probe, fixed, and studied byflow-cytometry and microscopy. Labeling was observed for all threeunnatural cyclooctyne analogues, with the relative labeling trackingwith the kinetics of the parent cyclooctynes. The labeling was shown tobe comparable to that used for TBTA plus the linear alkyneD-propargylglycine (alkDala), with the advantage that this labeling canoccur in the absence of copper. Microscopy showed that labeling waslimited to the cell wall, consistent with incorporation of the aminoacid into peptidoglycan.

To further confirm that peptidoglycan was being labeled, a competitionexperiment was performed. Incubation of L. monocytogenes withcyclooctyne D-amino acid in the presence of excess D-alanine resulted ina decrease in fluorescence after labeling. Additionally, listerialacking PBP5 also showed enhanced labeling over wild type. Thisenhancement is comparable for both the relatively small alkDala and thebulkier cyclooctyne amino acids, suggesting that the PBP5 enzyme istolerant of larger, unnatural substrates.

Example 2 Incorporation of Azide- and Alkyne-Modified D-Amino Acids intoPeptidoglycan

This disclosure describes a chemical approach for probing PG in vivo viametabolic labeling and bioorthogonal chemistry. A wide variety ofbacterial species incorporated azide and alkyne-functionalized D-alanineinto their cell walls, which we visualized by covalent reaction withclick chemistry probes. The D-alanine analogs were specificallyincorporated into nascent PG of the intracellular pathogen Listeriamonocytogenes both in vitro and during macrophage infection. Metabolicincorporation of D-alanine derivatives and click chemistry detectionconstitute a facile, modular platform that facilitates unprecedentedspatial and temporal resolution of PG dynamics in vivo.

Materials and Methods

In Vitro D-Alanine Derivative Labeling.

The origin and identity of bacterial strains are detailed in SupportingInformation. Alexa Fluor 350 succiminidyl-ester (Invitrogen) wasprepared exactly as described.(35) Vancomycin-BODIPY (Invitrogen) wasused as a 1:1 mixture with unlabeled vancomycin at a final concentrationof 1 μg/ml. D-alanine derivatives were used at 0.5-10 mM for labeling invitro and 5-50 mM for labeling in macrophages. Analysis of PG by HPLCand mass spectrometry is included in Supporting Information.Strain-promoted cycloaddition was performed using 1 μM or 10 μM ofBARAC(36) or DIFO(37) conjugates, respectively. CuAAC was performed oncell surfaces and lysates as described(38) using 20 μM of azide oralkyne conjugate. For cell surface labeling, we obtained the bestresults when bacteria were fixed with 2% formaldehyde prior to the clickreactions. The Staudinger ligation was performed by incubating celllysates in 500 μM phosphine-FLAG(39) overnight at 37° C.

Imaging Analysis.

Details on microscopy are provided in Supporting Information. Formulti-color images (FIG. 3), fluorescence intensities were adjusted inSLIDEBOOK software (Intelligent Imaging Innovations) to images of bothuntreated, non-fluorescent control cells as well as controls labeledwith a single reagent or fluorophore. Contrast was increased equallyacross the greyscale images in Image™ Images stacks for FIG. 3B weremade into a z-projection image using the average setting and deconvolvedin ImageJ using PSF generator and iterative deconvolver pluginsdeveloped by OptiNav, Inc. Separate channel images were merged togetherto form an RCSB composite. For single-color images (FIG. 1C),fluorescence intensities were normalized to controls lacking alkDala orazDala in SLIDEBOOK software.

In Vivo D-Alanine Derivative Labeling.

L. monocytogenes were grown overnight at 30° C. without shaking. Thenext day, the bacteria were washed in PBS then added to J774 cellsgrowing in chamber slides at a multiplicity of infection of 10. After 30min, the coculture was washed in PBS and incubated in fresh medium.Gentamicin was added after an additional 30 min and alkDala was addedeither 30 min or 3.5 h after addition of bacteria. After 4 h cells werewashed in PBS then incubated 3×5 min in fresh, pre-warmed mediumcontaining gentamicin but not D-alanine derivative. The coculture waswashed in PBS, fixed in 4% formaldehyde and permeabilized with 1%Triton-X. The CuAAC reaction was performed with azido-fluor 488 for 30min. The slides were mounted in Vectashield (Vector Labs) for imaging.

Results

Efforts were made to determine whether D-amino acids bearingbioorthogonal functional groups could be used for metabolic labeling.Azides and alkynes are small chemical reporters that are stable in andabsent from biological systems.(19) They undergo selective reaction witheach other and, in the case of the azide, with phosphines and strainedcyclooctynes as well.(19) To evaluate the ability of unnatural D-alaninederivatives to access the cell wall, bacteria were grown in mediacontaining R-propargylglycine (compound 1, FIG. 1B, abbreviated alkDala)or R-2-amino-3-azidopropanoic acid (compound 2, FIG. 1B, abbreviatedazDala). The cells were then reacted with a complementary fluorescentdye using strain-promoted cycloaddition or copper-catalyzed azide-alkynecycloaddition (CuAAC)(19) (FIG. 1B). Clear cell surface labeling of allspecies tested (FIG. 1C), including several Gram-positive bacteria, oneGram-negative and Mycobacterium tuberculosis, a Gram-positive with anunusually complex cell wall, was observed by microscopy. Theconcentrations of D-alanine analog used for labeling did not inhibitbacterial growth.

The studies focused on the facultative intracellular pathogen L.monocytogenes for further characterization of D-alanine analogmetabolism AlkDala labeling of wildtype L. monocytogenes was compared tothat of a D-alanine auxotroph (termed dal⁻ dat⁻).(20) The auxotroph,which is unable to synthesize natural D-alanine as an endogenouscompetitor of the synthetic substrate, labeled more strongly (FIG. 1D).Similarly, addition of exogenous D-alanine suppressed azDala labeling(FIG. 1E). These results suggest that alkDala and azDala access the samemetabolic pathways as natural D-alanine.

FIGS. 1A-E.

Incubation of bacteria in D-alanine analogs followed by reaction withclick chemistry probes results in cell surface fluorescence. (A)Chemical structure of Escherchia coli and Listeria monocytogenes PG(mDAP=meso-diaminopimelic acid). Newly synthesized disaccharidepentapeptides are substrates for penicillin-binding protein (PBP)processing, including cross-linking by transpeptidases (TPases) andtrimming by carboxypeptidases (CPases). (B) Schematic representation ofin vitro metabolic labeling with D-alanine analogs (1, 2) followed byclick chemistry detection (3-6). R-propargylglycine (1, alkDala),2-amino-3-azidopropanoic acid (2, azDala), azide (3) and alkyne (4)conjugates for Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC),cyclooctyne probe for strain-promoted cycloaddition (5), phosphinereagent for Staudinger ligation (6). The identity of the green starvaries according to application e.g. fluorophores or affinity handles.(C) Clockwise from top left: E. coli (Ec), Bacillus subtilis (Bs), L.monocytogenes (Lm), Mycobacterium tuberculosis (Mt), Streptomycescoelicolor (Sc), Corynebacterium glutamicum (Cg). Scale bars, 1 μm. (D)The dal⁻ dat⁻ D-alanine auxotroph labels better than wildtype L.monocytogenes with alkDala, CuAAC. MFI, mean fluorescence intensity. (E)2 mM D- but not L-alanine competes labeling by 5 mM azDala,strain-promoted cycloaddition in dal- dat- L. monocytogenes. The mutantwas supplemented with an additional 1 mM D-alanine in all conditions for(E). Error bars for (D) and (E), +/−s.d. *P=0.0002 for (D), *P=2×10⁻⁵for (E), two-tailed Student's t tests. Data are in triplicate andrepresentative of four and two experiments, respectively.

It was hypothesized that there are three potential sites of D-alanineanalog incorporation: proteins, lipoteichoic acids (LTA) and PG. Thefirst would likely require both racemization and a highly promiscuousaminoacyl tRNA synthetase.(21, 22) To address this possibility directly,lysates from azDala-treated L. monocytogenes were reacted withalkyne-biotin and analyzed the products by immunoblot. No azide-labeledproteins were detected. Although D-alanine incorporation systems for LTAare generally very specific,(23) the second theoretical possibility thatD-alanine analogs might label this biopolymer was addressed. LTAenriched from azDala-treated L. monocytogenes was reacted withphosphine-FLAG and probed by immunoblot as above. No azide-labeledspecies was detected in these cell wall preparations. Furthermore, amutant that does not produce LTA(24) labeled with identical efficiencyto wildtype bacteria. These data suggest that the D-alanine derivativesdo not incorporate into proteins or LTA. D-alanylation of the other L.monocytogenes teichoic acid polymer, wall teichoic acid (WTA), has notbeen observed.(25, 26)

Having ruled out other potential sites of D-alanine derivative labeling,direct evidence of its incorporation into PG was sought. PG comprises arepeating disaccharide to which is conjugated a short peptide, termedthe stem peptide (FIG. 1A). Although newly synthesized PG stem peptideusually terminates in D-alanine-D-alanine, diverse bacterial phylaproduce and incorporate D-amino acids other than D-alanine into thosepositions. (17, 18) The process is flexible; various natural andunnatural D-amino acids appear to incorporate, albeit at varyingefficiencies.(16-18) To test whether D-alanine derivatives incorporateinto PG, Escherchia coli or L. monocytogenes was incubated with alkDala;afterwards, the cells were reacted with azido-fluor 488, and PG waspurified from the cells for further analysis. After digesting the PGwith muramidase, HPLC was used to detect muropeptides by absorbance ateither 204 nm (to visualize all species) or 500 nm (to identifyfluorophore-containing fragments). Finally the most abundant peaks at500 nm were collected, and mass spectrometry was used to assign theirchemical structures. This analysis showed that alkDala inserts into thefourth position of the stem peptide in E. coli PG and the fifth positionin L. monocytogenes PG (FIG. 2). PG samples from E. coli incubated inazDala alone were analyzed; and it was determined that the fraction ofD-alanine that had been replaced with the synthetic analog was roughly50% of the tetrapeptide pool and 15% of the total muropeptidepopulation. Importantly, even long periods of alkDala incubation did notappreciably change PG structure compared to D-alanine incubationperformed in parallel.

FIGS. 2A-D.

AlkDala incorporates into L. monocytogenes PG. (A) HPLC chromatograms ofnon-reduced muropeptides from L. monocytogenes incubated in the presenceof 5 mM D-alanine (left) or alkDala (right) then reacted withazido-fluor 488. Absorbance at 204 nm, blue, and at 500 nm, red, areshown. The trace for the alkDala-treated sample is enlarged in (C). Themost abundant peaks detected at 500 nm were collected and subjected toanalysis by mass spectrometry, (D), to identify the chemical structureof the alkDala-containing muropeptides conjugated to azido-fluor 488,(B).

The positional selectivity of alkDala in PG implies a biosyntheticpathway of incorporation. There are two primary mechanisms for insertionof D-amino acids into PG: periplasmic editing of the mature polymer andcytosolic incorporation into PG precursors.(18, 27) The first process isan L,D- or D,D-transpeptidation reaction that, respectively, results ina new D-amino acid at the fourth or fifth position of the PG stempeptide. The second process is catalyzed by intracellular ligases andresults in a new D-amino acid only at the fifth position. Thepentapeptide substrates that support D,D-transpeptidase incorporation ofD-amino acids in other bacteria are short-lived in L. monocytogenesbecause they are rapidly lost during PG maturation (FIG. 1A,(28-30)).Thus the observation of alkDala in the fifth position suggests thatD-alanine analogs incorporate into newly synthesized L. monocytogenesPG. Three additional lines of evidence support this notion. First,D-alanine analog labeling was greatest at the peak of new cell wallproduction, in exponential phase growth. Next, incubating L.monocytogenes in alkDala for one generation followed by reaction withazido-fluor 545 resulted in signal that colocalized with that ofvancomycin-BODIPY, a marker of nascent PG (FIG. 3A,(8)). Finally,treatment of bacteria with fosfomycin, a drug that inhibits PG synthesisvery early in the pathway, completely abrogated alkDala labeling,whereas treatment with penicillin and meropenem, antibiotics that targetperiplasmic editing enzymes, had a much weaker effect.

L. monocytogenes naturally infects macrophages where it can escape fromthe phagosome and proliferate in the cytosol. The dal⁻ dat⁻ D-alanineauxotroph shows wildtype infectivity in cultured cells when D-alanine isadded to the tissue culture medium.(20) This observation suggests thatD-alanine and perhaps other D-amino acids are effectively taken up bymacrophages at levels sufficient to support L. monocytogenes growth.Moreover, because eukaryotic cells do not generally produce D-aminoacids, it was reasoned that D-alanine analogs might selectively labelbacteria inside of host cells. To test this hypothesis, J774 macrophageswere infected with L. monocytogenes, removed extracellular bacteria andtreated the coculture with alkDala. Cells were incubated in alkDala forless than one L. monocytogenes generation, to label newer PG, or forseveral generations, to label both new and mature PG. Chemical methods,used previously for labeling intracellular proteins in mammalian cells(31, 32), were adapted to visualize PG by reaction with azido-fluor 488.Alkyne-dependent signal varied according to alkDala incubation time: L.monocytogenes labeled more at the septa when incubated for a short pulseand at the septa and poles when labeled for a longer one (FIG. 3B). Thespatial distribution of fluorescence approximated that observed onbacteria grown alone in vitro (FIG. 1C, top right).

FIGS. 3A-C.

AlkDala labels newly synthesized L. monocytogenes PG in vitro and invivo. (A) Fluorescent signals from vancomycin-BODIPY (vanc-fl) andalkDala, azido-fluor 545 (az-fl) colocalize. L. monocytogenes wereincubated in Alexa Fluor 350 succiminidyl-ester (NHS-fl) tonon-specifically label cell wall proteins then washed and resuspended inalkDala for 1 hr and vanc-fl for the last 30 min. (B), (C) AlkDalalabels intracellular L. monocytogenes. J774 cells were infected with L.monocytogenes bearing red fluorescent protein (RFP) under the actApromoter (34). Because this promoter is regulated by PrfA and inducedupon escape from the phagosome, RFP expression correlates with entryinto the cytosol (34). Extracellular bacteria were washed away after 30min and the infected cells were incubated in fresh medium containinggentamicin. 50 mM alkDala was added for the remaining 3.5 hrs (C) or forthe last 30 minutes only (B). Infected cells were fixed, permeabilizedand reacted with azido-fluor 488. Top rows, fluorescent images, bottomrows, fluorescent and brightfield merge.

Although the method was developed using L. monocytogenes, the D-alanineanalogs incorporate into a wide variety of bacteria and will promoteinvestigation of diverse PG dynamics both in vitro and during infection.Furthermore, D-alanine analogs will greatly expand the scope of PGanalysis. Judicious use of the ever-expanding azide and alkyne-reactiveprobe kit should permit integration of biochemical, genetic and cellbiological data that historically have been collected and analyzed inisolation. Multi-tiered interrogation of PG in the host environment mayuncover new avenues for inhibiting this well-validated drug target.

FIG. 4 depicts labeling of C. glutamicum grown in the presence orabsence of 20 mM norbornene-D-alanine (norDala) for three doublings,washed then incubated in 10 uM tetrazine-fluorescein for 1 hr prior toimaging. FIG. 4B, C. glutamicum grown in the presence or absence of 5 mMor 20 mM norDala, detected as above then subjected to flow cytometry toquantitate fluorescence intensity. MFI, mean fluorescence intensity.Error bars, +/−s.d.

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While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. An isolated, modified peptidoglycan comprising atleast one modified D-amino acid comprising a bioorthogonal group.
 2. Anisolated, modified peptidoglycan comprising at least one modifiedD-amino acid chemically conjugated to a reagent via a heterocycle,wherein the modified D-amino acid is an azide-modified, analkyne-modified, or a norbornene-modified D-amino acid, and the reagentis an azide-containing reagent, an alkynyl reagent or anorbornene-reactive reagent.
 3. The isolated, modified PG of claim 2,wherein the reagent comprises a detectable label.
 4. The isolated,modified PG of claim 2, wherein the reagent comprises a therapeuticagent.
 5. The isolated, modified PG of claim 2, wherein the reagentcomprises an immunomodulatory molecule.
 6. A modified bacterial cellcomprising a modified peptidoglycan, wherein the modified peptidoglycancomprises at least one modified D-amino acid, wherein the at least onemodified D-amino acid is an azide-modified, an alkyne-modified, or anorbornene-modified D-amino acid.
 7. A method of identifying an agentthat inhibits peptidoglycan synthesis, the method comprising: a)contacting a bacterial cell in vitro with a test agent; and b)determining the effect, if any, of the test agent on incorporation of amodified D-amino acid into peptidoglycan in the bacterial cell; whereina test agent that inhibits incorporation of the modified D-amino acidinto peptidoglycan in the bacterial cell is considered a candidate agentfor inhibiting peptidoglycan synthesis.
 8. The method of claim 7,wherein the bacterial cell is present intracellularly in a eukaryotichost cell.
 9. The method of claim 7, wherein the eukaryotic host cell isa macrophage.
 10. The method of claim 7, wherein incorporation of themodified D-amino acid into the peptidoglycan is determined by contactingthe cell with a reagent selected from a detectably labeledazide-containing reagent, a detectably labeled alkynyl reagent and adetectably labeled norbornene-reactive reagent.
 11. The method of claim7, wherein the detectable label is a fluorescent label.
 12. Acomposition comprising: a) an isolated, modified peptidoglycancomprising at least one modified D-amino acid, wherein the modifiedD-amino acid is chemically conjugated to a reagent selected from anazide-containing reagent, an alkynyl reagent and a norbornene-reactivereagent, and the isolated, modified peptidoglycan is described by theformula:PG-L¹-Z-L²-Y wherein: PG is the modified peptidoglycan, L¹ and L² areindependently optional linkers, Z is a heterocycle, and Y is atherapeutic agent or an immunomodulatory molecule; and b) apharmaceutically acceptable excipient.
 13. The composition of claim 12,wherein Y is a therapeutic agent.
 14. The composition of claim 12,wherein Y is an immunomodulatory molecule.
 15. The composition of claim14, wherein Y is an antigen.
 16. A method of modulating an immuneresponse in an individual, the method comprising administering to theindividual an effective amount of a composition of claim 12.